CN104961825A

CN104961825A - RSV-specific binding molecules and means for producing them

Info

Publication number: CN104961825A
Application number: CN201410587255.2A
Authority: CN
Inventors: H·斯皮茨; T·博蒙特; M·J·卡瓦肯博; E·雅苏达
Original assignee: MedImmune Vaccines Inc
Current assignee: MedImmune Ltd; MedImmune Vaccines Inc
Priority date: 2007-06-01
Filing date: 2008-05-30
Publication date: 2015-10-07
Also published as: US20200325213A1; US20160244509A1; SI2170952T1; US20140377279A9; WO2008147196A2; US8562996B2; IL202288A; PT2170952E; ES2575129T3; US20140072575A1; NZ621824A; HRP20160632T1; KR101652652B1; NZ712667A; BRPI0812878A2; AU2008257801B8; EP2578600A2; CY1117767T1; US10059757B2; DK2170952T3

Abstract

The invention provides antibodies and functional equivalents thereof which are capable of specifically binding RSV, and means and methods for producing them.

Description

RSV specific binding molecules and their mode of generation

The application is the CN 200880023301.9 that 2008.05.30 submits to, is entitled as the divisional application of " RSV specific binding molecules and their mode of generation ".

The present invention relates to biology and medical field.

Respiratory syncytial virus (RSV) is common common cold virus, belongs to Paramyxoviridae.RSV has virulence, easily propagates, and be the lower respiratory illness of less than 2 years old children most commonly encountered diseases because of.In RSV season, the day care children up to 98% are by infected.In the children of infection RSV, 0.5% to 3.2% needs hospital care.In the U.S., about have 90 every year, 000 example is sought medical advice and 4500 example death.Because RSV causes the major risk factors of hospital care to be the children in below 6 week age of premature labor, chronic lung disease, congenital heart disease, immunological inadequacy and other aspect health.The positive bronchiolitis of RSV is not effectively treated, the Supportive Care of enough nutrition and oxygen therapy form can only be carried out.Antiviral therapy is as invalid in rsv infection in ribavirin.Beautiful pearl monoclonal antibody (also referred to as the Synagis) registration of a kind of monoclonal antibody handkerchief is for preventing rsv infection.The beautiful pearl monoclonal antibody of handkerchief is the monoclonal antibody of the RSV fusion rotein of genetic modification (humanization).But handkerchief beautiful pearl monoclonal antibody is always ineffective.Therefore, this area needs other antibody and treatment to resist RSV.

The object of this invention is to provide mode and the method for antagonism and/or prevention RSV relative disease.Another object of the present invention is to provide RSV antibody or its function equivalent of alternative and/or improvement, and provides the stabilized cell that can produce RSV antibody or its function equivalent.

The invention provides can the antibody of specific binding RSV and its function equivalent.This antibody-like and/or function equivalent are in this article also referred to as " anti-RSV antibodies " or " RSV-specific antibody ", and they can at least one component of specific binding RSV, certain epi-position of such as rsv protein matter.Term " specific binding " does not comprise non-specific adhesion.Anti-RSV antibodies of the present invention and function equivalent are particularly suitable for the detrimentally affect resisting and/or prevent at least partly rsv infection and/or rsv infection.The particularly preferred anti-RSV antibodies of one of the present invention is the antibody being called " D25 ", and it has heavy chain district as shown in Figure 11 A-D and light chain district.The CDR sequence of the D25 of the antigenic binding property of concrete generation D25 is shown in Figure 11 D.Compared with the beautiful pearl monoclonal antibody of registered anti-RSV antibodies handkerchief, antibody D25 seems to have excellent performance (Fig. 8).Such as, the IC50 value of D25 in the external Neutralizing test of HEp-2 cell infection RSV is about 0.4-1.5ng/ml, and the IC50 value of the beautiful pearl monoclonal antibody of handkerchief is about 453ng/ml.

In this article, the function equivalent of antibody is defined as the Functional portions of antibody, derivative or analogue.

The Functional portions of antibody is defined as the part with described antibody with at least one identical characteristics (kind is identical, and amount is not necessarily identical).Described Functional portions can with described antibodies same antigen, but combination degree is not necessarily identical.The Functional portions of antibody preferably includes single domain antibody, single-chain antibody, single chain variable fragment (scFv), Fab fragment or F (ab') ₂fragment.

The functional derivatives of antibody is defined as that to obtain at least one Te – preferred antigens of compound through house of correction substantially identical in conjunction with Te – kind, and the antibody that amount is not necessarily identical.In many ways, such as replaced by conservative amino acid and provide derivative, wherein amino-acid residue is replaced by another residue of character (size, hydrophobicity etc.) basic simlarity, such that general function is unlikely to be had a strong impact on.

Those skilled in the art can produce the similar compound of antibody.This operation is carried out by screening peptide library or phage display library.This analogue has at least one kind substantially with described antibody identical and measure not necessarily identical characteristic.

As well known to the skilled person, heavy chain of antibody forms that type larger in two kinds of chain types of immunoglobulin molecules.Heavy chain comprises constant region and variable region, and wherein variable region participates in antigen combination.The light chain of antibody forms that type less in two kinds of chain types of immunoglobulin molecules.Light chain comprises constant region and variable region.Variable region participates in antigen and combines together with variable region of heavy chain.

Complementary determining region (CDR) is the hypervariable region in variable region of heavy chain and variable region of light chain.The heavy chain of antibody forms antigen binding site together with the CDR of the light chain of connection.

Since the present invention recognizes that the sequence of CDR shown in Figure 11 provides required RSV binding characteristic, technician just can produce the variant comprising the CDR sequence that at least one changes.Such as, apply conservative amino acid to replace.Conservative amino acid replaces another kind of aminoacid replacement one seed amino acid comprised with character (size, hydrophobicity etc.) basic simlarity, such that general function is unlikely to be had a strong impact on.

Also at least one CDR sequence shown in Figure 11 may be changed, to produce antibody variants or its function equivalent of at least one characteristic changing compared with D25.Preferably, the antibody provided or function equivalent comprise the CDR sequence identical with the sequence of CDR shown in Figure 11 at least 70%, to keep, even to improve the favourable binding characteristic of D25 at least partly.CDR sequence shown in preferred change Figure 11, the characteristic comprising at least one to make the antibody that obtains or function equivalent compared with D25 and improve, the binding affinity such as improved, selectivity and/or stability.Therefore, the antibody variants or its function equivalent that comprise the aminoacid sequence identical with the sequence of CDR shown in Figure 11 at least 70% belong to the scope of the invention.This area can obtain the method for various change aminoacid sequence.Such as, synthetic has heavy chain or the sequence of light chain of required CDR sequence.Preferably, utilize (such as) random or site-directed mutagenesis that the nucleotide sequence of coding CDR is suddenlyd change.

Therefore, the present invention provide in first a kind of separation, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its function equivalent, it comprises:

-heavy chain CDR1 sequence, it comprises the sequence identical with sequence NYIIN at least 70%, and/or

-heavy chain CDR2 sequence, it comprises the sequence identical with sequence GIIPVLGTVHYAPKFQG at least 75%, and/or

-heavy chain CDR3 sequence, it comprises the sequence identical with sequence ETALVVSTTYLPHYFDN at least 70%, and/or

-light chain CDR1 sequence, it comprises the sequence identical with sequence QASQDIVNYLN at least 85%, and/or

-light chain CDR2 sequence, it comprises the sequence identical with sequence VASNLET at least 70%.

Preferably, described antibody also comprises light chain CDR3 sequence, and this sequence contains the sequence identical with sequence QQYDNLP at least 70%.

Preferably, antibody of the present invention or function equivalent comprise and at least one CDR sequence at least 75% shown in Figure 11 D, and more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical CDR sequence.Most preferably, antibody of the present invention or function equivalent comprise the CDR sequence identical with at least one CDR sequence at least 95% shown in Figure 11 D.Above-mentioned particularly preferred antibody D25 comprises the CDR sequence be made up of CDR sequence shown in Figure 11 D.Therefore, the particularly preferred embodiment of the present invention provide a kind of separation, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its function equivalent, it comprises:

-heavy chain CDR1 sequence, it comprises sequence NYIIN, and/or

-heavy chain CDR2 sequence, it comprises sequence GIIPVLGTVHYAPKFQG, and/or

-heavy chain CDR3 sequence, it comprises sequence ETALVVSTTYLPHYFDN, and/or

-light chain CDR1 sequence, it comprises sequence QASQDIVNYLN, and/or

-light chain CDR2 sequence, it comprises sequence VASNLET.

Preferably, described antibody also comprises the light chain CDR3 sequence containing sequence QQYDNLP.

In one embodiment, the antibody provided or function equivalent comprise three heavy CDR sequences as shown in Figure 11 D and three CDR sequence, or with its at least 70%, preferably at least 80%, more preferably at least 85% identical sequence.Therefore, there is provided a kind of to be further separated, the antibody of synthesis or restructuring or its function equivalent, it comprises the heavy chain CDR1 sequence containing the sequence identical with sequence NYIIN at least 70%, heavy chain CDR2 sequence containing the sequence identical with sequence GIIPVLGTVHYAPKFQG at least 70%, heavy chain CDR3 sequence containing the sequence identical with sequence ETALVVSTTYLPHYFDN at least 70%, light chain CDR1 sequence containing the sequence identical with sequence QASQDIVNYLN at least 70%, light chain CDR2 sequence containing the sequence identical with sequence VASNLET at least 70% and the light chain CDR3 sequence containing the sequence identical with sequence QQYDNLP at least 70%.Described antibody or function equivalent preferably comprise with the heavy CDR sequences shown in Figure 11 D and CDR sequence at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical CDR sequence.Also providing package contains antibody or the function equivalent of above-mentioned heavy chain CDR1, CDR2 and CDR3 sequence and above-mentioned light chain CDR1, CDR2 and CDR3 sequence.

Also providing package contains antibody or its function equivalent of the heavy chain variable amino acid sequence identical with sequence of heavy chain at least 70% shown in Figure 11.This kind of sequence of heavy chain provides required RSV binding characteristic, as antibody D25 prove.Therefore, also provide a kind of antibody or its function equivalent, its sequence of heavy chain comprises the sequence identical with sequence QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLG TVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHY FDNWGQGTLVTVSS at least 70%.And the chain variable region amino acid sequence identical with sequence of light chain at least 70% shown in Figure 11 provides required RSV binding characteristic, as antibody D25 prove.Therefore, also provide a kind of antibody or its function equivalent, it has the sequence of light chain identical with sequence D IQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETG VPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTV at least 70%.Antibody of the present invention or Functional portions preferably comprise with sequence of heavy chain shown in Figure 11 and/or sequence of light chain at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical weight chain variabl area sequence and/or light-chain variable sequence.Homology is higher, and described antibody or Functional portions are more close to antibody D25.Antibody of the present invention or Functional portions preferably comprise the heavy chain of similar D25 and the heavy chain of light chain and light chain.Therefore, a kind of antibody or Functional portions are also provided, its comprise with sequence of heavy chain shown in Figure 11 and sequence of light chain at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical sequence of heavy chain and sequence of light chain.

An embodiment provides a kind of antibody or its function equivalent, and it comprises the sequence of light chain of sequence of heavy chain that sequence of heavy chain as shown in Figure 11 forms and sequence of light chain formation as shown in Figure 11.Or, as well known to the skilled person, may produce and shorten but maintain heavy chain or the sequence of light chain of interested binding characteristic.Preferably, compared with initial heavy chain or light chain, heavy chain or the light chain of this shortening of generation have shorter constant region.Preferred maintenance variable region is constant.Such as, Fab fragment or F (ab') is produced based on sequence of heavy chain shown in Figure 11 or sequence of light chain ₂fragment.Therefore, the antibody function equivalent of the Functional portions at least comprising sequence shown in Figure 11 is also provided.The length of described Functional portions is at least 20 amino acid, comprise the sequence identical with the sequence of heavy chain CDR1 shown in Figure 11 D at least 70%, and/or the sequence identical with the sequence of heavy chain CDR2 shown in Figure 11 D at least 75%, and/or the sequence identical with the sequence of heavy chain CDR3 shown in Figure 11 D at least 70%, and/or the sequence identical with the sequence of light chain CDR1 shown in Figure 11 D at least 85%, and/or the sequence identical with the sequence of light chain CDR2 shown in Figure 11 D at least 70%.Preferably, described Functional portions also comprises the sequence identical with the sequence of light chain CDR3 shown in Figure 11 D at least 70%.

The particularly preferred anti-RSV antibodies of another kind of the present invention is the antibody being called " AM14 ", and it has heavy chain district as shown in Figure 14 A and light chain district.The CDR sequence of the AM14 of the antigenic binding property of concrete generation AM14 is also see Figure 14 A.

Since the present invention recognizes that CDR sequence shown in Figure 14 A provides required RSV binding characteristic, technician just can produce the variant comprising the CDR sequence that at least one changes.Such as, apply conservative amino acid to replace.Conservative amino acid replaces another kind of aminoacid replacement one seed amino acid comprised with character (size, hydrophobicity etc.) basic simlarity, such that general function is unlikely to be had a strong impact on.

Also at least one CDR sequence shown in Figure 14 A may be changed, to produce antibody variants or its function equivalent of at least one characteristic changing compared with AM14.Preferably, the antibody provided or function equivalent comprise the CDR sequence identical with CDR sequence at least 70% shown in Figure 14 A, to keep, even to improve the favourable binding characteristic of AM14 at least partly.CDR sequence shown in preferred change Figure 14 A, the characteristic comprising at least one to make the antibody that obtains or function equivalent compared with AM14 and improve, the binding affinity such as improved, selectivity and/or stability.Therefore, the antibody variants or its function equivalent that comprise the aminoacid sequence identical with CDR sequence at least 70% shown in Figure 14 A belong to the scope of the invention.This area can obtain the method for various change aminoacid sequence.Such as, synthetic has heavy chain or the sequence of light chain of required CDR sequence.Preferably, utilize (such as) random or site-directed mutagenesis that the nucleotide sequence of coding CDR is suddenlyd change.

Therefore, the present invention provide in one aspect a kind of separation, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its Functional portions, derivative and/or analogue, it comprises:

-heavy chain CDR1 sequence, it comprises the sequence identical with sequence GFSFSHYA at least 70%, and/or

-heavy chain CDR2 sequence, it comprises the sequence identical with sequence ISYDGENT at least 70%, and/or

-heavy chain CDR3 sequence, it comprises the sequence identical with sequence A RDRIVDDYYYYGMDV at least 70%, and/or

-light chain CDR1 sequence, it comprises the sequence identical with sequence QDIKKY at least 70%, and/or

-light chain CDR2 sequence, it comprises the sequence identical with sequence D AS at least 70%, and/or

-light chain CDR3 sequence, it comprises the sequence identical with sequence QQYDNLPPLT at least 70%.

Preferably, antibody of the present invention or function equivalent comprise and at least one CDR sequence at least 75% shown in Figure 14 A, and more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical CDR sequence.Most preferably, antibody of the present invention or function equivalent comprise the CDR sequence identical with at least one CDR sequence at least 95% shown in Figure 14 A.Above-mentioned particularly preferred antibody A M14 comprises the CDR sequence be made up of CDR sequence shown in Figure 14 A.Therefore, the particularly preferred embodiment of the present invention provide a kind of separation, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its function equivalent, it comprises:

-heavy chain CDR1 sequence, it comprises sequence GFSFSHYA, and/or

-heavy chain CDR2 sequence, it comprises sequence ISYDGENT, and/or

-heavy chain CDR3 sequence, it comprises sequence A RDRIVDDYYYYGMDV, and/or

-light chain CDR1 sequence, it comprises sequence QDIKKY, and/or

-light chain CDR2 sequence, it comprises sequence D AS, and/or

-light chain CDR3 sequence, it comprises sequence QQYDNLPPLT.

In one embodiment, the antibody provided or function equivalent comprise three heavy CDR sequences as shown in Figure 14 A and three CDR sequence, or with its at least 70% identical sequence.Therefore, there is provided a kind of to be further separated, the antibody of synthesis or restructuring or its function equivalent, it comprises the heavy chain CDR1 sequence containing the sequence identical with sequence GFSFSHYA at least 70%, heavy chain CDR2 sequence containing the sequence identical with sequence ISYDGENT at least 70%, heavy chain CDR3 sequence containing the sequence identical with sequence A RDRIVDDYYYYGMDV at least 70%, light chain CDR1 sequence containing the sequence identical with sequence QDIKKY at least 70%, light chain CDR2 sequence containing the sequence identical with sequence D AS at least 70% and the light chain CDR3 sequence containing the sequence identical with sequence QQYDNLPPLT at least 70%.Described antibody or function equivalent preferably comprise with the heavy CDR sequences shown in Figure 14 A and CDR sequence at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical CDR sequence.Also providing package contains antibody or the function equivalent of heavy chain CDR1, CDR2 and CDR3 sequence of above-mentioned Figure 14 A and light chain CDR1, CDR2 and CDR3 sequence of above-mentioned Figure 14 A.

Also providing package contains antibody or its function equivalent of the heavy chain amino acid sequence identical with sequence of heavy chain at least 70% shown in Figure 14 A.This kind of sequence of heavy chain provides required RSV binding characteristic, as antibody A M14 prove.Therefore, also provide a kind of antibody or its function equivalent, its sequence of heavy chain comprises the sequence identical with sequence EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGE NTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDV WGQGATVTVSS at least 70%.And the light-chain amino acid sequence identical with sequence of light chain at least 70% shown in Figure 14 A provides required RSV binding characteristic, as antibody A M14 prove.Therefore, also provide a kind of antibody or its function equivalent, it has the sequence of light chain identical with sequence D IQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETG VPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGGTKVEIKRTV at least 70%.Antibody of the present invention or Functional portions preferably comprise with sequence of heavy chain shown in Figure 14 A and/or sequence of light chain at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical weight chain variabl area sequence and/or light-chain variable sequence.Homology is higher, and described antibody or Functional portions are more close to antibody A M14.Antibody of the present invention or Functional portions preferably comprise the heavy chain of similar AM14 and the heavy chain of light chain and light chain.Therefore, a kind of antibody or Functional portions are also provided, its comprise with sequence of heavy chain shown in Figure 14 A and sequence of light chain at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical sequence of heavy chain and sequence of light chain.

An embodiment provides a kind of antibody or its function equivalent, and it comprises the sequence of heavy chain be made up of sequence of heavy chain shown in Figure 14 A and the sequence of light chain be made up of sequence of light chain shown in Figure 14 A.Or, as well known to the skilled person, may produce and shorten but maintain heavy chain or the sequence of light chain of interested binding characteristic.Preferably, compared with initial heavy chain or light chain, heavy chain or the light chain of this shortening of generation have shorter constant region.Preferred maintenance variable region is constant.Such as, Fab fragment or F (ab') is produced based on sequence of heavy chain shown in Figure 14 A or sequence of light chain ₂fragment.Therefore, the antibody function equivalent of the Functional portions at least comprising sequence shown in Figure 14 A is also provided.The length of described Functional portions is at least 20 amino acid, comprises the sequence identical with at least one CDR sequence at least 70% shown in Figure 14 A.

The particularly preferred anti-RSV antibodies of another kind of the present invention is the antibody being called " AM16 ", and it has heavy chain district as shown in Figure 14B and light chain district.The CDR sequence of the AM16 of the antigenic binding property of concrete generation AM16 is also see Figure 14 B.

Since the present invention recognizes that CDR sequence shown in Figure 14 B provides required RSV binding characteristic, technician just can produce the variant comprising the CDR sequence that at least one changes.Such as, apply conservative amino acid to replace.Conservative amino acid replaces another kind of aminoacid replacement one seed amino acid comprised with character (size, hydrophobicity etc.) basic simlarity, such that general function is unlikely to be had a strong impact on.

Also at least one CDR sequence shown in Figure 14 B may be changed, to produce antibody variants or its function equivalent of at least one characteristic changing compared with AM16.Preferably, the antibody provided or function equivalent comprise the CDR sequence identical with CDR sequence at least 70% shown in Figure 14 B, to keep, even to improve the favourable binding characteristic of AM16 at least partly.CDR sequence shown in preferred change Figure 14 B, the characteristic comprising at least one to make the antibody that obtains or function equivalent compared with AM16 and improve, the binding affinity such as improved, selectivity and/or stability.Therefore, the antibody variants or its function equivalent that comprise the aminoacid sequence identical with CDR sequence at least 70% shown in Figure 14 B belong to the scope of the invention.This area can obtain the method for various change aminoacid sequence.Such as, synthetic has heavy chain or the sequence of light chain of required CDR sequence.Preferably, utilize (such as) random or site-directed mutagenesis that the nucleotide sequence of coding CDR is suddenlyd change.

-heavy chain CDR1 sequence, it comprises the sequence identical with sequence GFTFSSYN at least 70%, and/or

-heavy chain CDR2 sequence, it comprises the sequence identical with sequence ISAGSSYI at least 70%, and/or

-heavy chain CDR3 sequence, it comprises the sequence identical with sequence A REDYGPGNYYSPNWFDP at least 70%, and/or

-light chain CDR1 sequence, it comprises the sequence identical with sequence SSNIGAGYD at least 70%, and/or

-light chain CDR2 sequence, it comprises the sequence identical with sequence GNT at least 70%, and/or

-light chain CDR3 sequence, it comprises the sequence identical with sequence HSYDRSLSG at least 70%.

Preferably, antibody of the present invention or function equivalent comprise and at least one CDR sequence at least 75% shown in Figure 14 B, and more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical CDR sequence.Most preferably, antibody of the present invention or function equivalent comprise the CDR sequence identical with at least one CDR sequence at least 95% shown in Figure 14 B.Above-mentioned particularly preferred antibody A M16 comprises the CDR sequence be made up of CDR sequence shown in Figure 14 B.Therefore, the particularly preferred embodiment of the present invention provide a kind of separation, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its function equivalent, it comprises:

-heavy chain CDR1 sequence, it comprises sequence GFTFSSYN, and/or

-heavy chain CDR2 sequence, it comprises sequence ISAGSSYI, and/or

-heavy chain CDR3 sequence, it comprises sequence A REDYGPGNYYSPNWFDP, and/or

-light chain CDR1 sequence, it comprises sequence SSNIGAGYD, and/or

-light chain CDR2 sequence, it comprises sequence GNT, and/or

-light chain CDR3 sequence, it comprises sequence HSYDRSLSG.

In one embodiment, the antibody provided or function equivalent comprise three heavy CDR sequences as shown in Figure 14B and three CDR sequence, or with its at least 70% identical sequence.Therefore, there is provided a kind of to be further separated, the antibody of synthesis or restructuring or its function equivalent, it comprises the heavy chain CDR1 sequence containing the sequence identical with sequence GFTFSSYN at least 70%, heavy chain CDR2 sequence containing the sequence identical with sequence ISAGSSYI at least 70%, heavy chain CDR3 sequence containing the sequence identical with sequence A REDYGPGNYYSPNWFDP at least 70%, light chain CDR1 sequence containing the sequence identical with sequence SSNIGAGYD at least 70%, light chain CDR2 sequence containing the sequence identical with sequence GNT at least 70% and the light chain CDR3 sequence containing the sequence identical with sequence HSYDRSLSG at least 70%.Described antibody or function equivalent preferably comprise with the above-mentioned heavy CDR sequences shown in Figure 14 B and above-mentioned CDR sequence at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical CDR sequence.Also providing package contains antibody or the function equivalent of heavy chain CDR1, CDR2 and CDR3 sequence of above-mentioned Figure 14 B and light chain CDR1, CDR2 and CDR3 sequence of above-mentioned Figure 14 B.

Also providing package contains antibody or its function equivalent of the heavy chain amino acid sequence identical with sequence of heavy chain at least 70% shown in Figure 14 B.This kind of sequence of heavy chain provides required RSV binding characteristic, as antibody A M16 prove.Therefore, also provide a kind of antibody or its function equivalent, its sequence of heavy chain comprises the sequence identical with sequence EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHISAGSS YIYYSDSVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNWF DPWGQGTLVTVSS at least 70%.And the light-chain amino acid sequence identical with sequence of light chain at least 70% shown in Figure 14 B provides required RSV binding characteristic, as antibody A M16 prove.Therefore, also provide a kind of antibody or its function equivalent, it has the sequence of light chain identical with sequence QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNR PSGVSDRFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGSVFGGGTKLTV at least 70%.Antibody of the present invention or Functional portions preferably comprise with sequence of heavy chain shown in Figure 14 B and/or sequence of light chain at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical weight chain variabl area sequence and/or light-chain variable sequence.Homology is higher, and described antibody or Functional portions are more close to antibody A M16.Antibody of the present invention or Functional portions preferably comprise the heavy chain of similar AM16 and the heavy chain of light chain and light chain.Therefore, a kind of antibody or Functional portions are also provided, its comprise with sequence of heavy chain shown in Figure 14 B and sequence of light chain at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical sequence of heavy chain and sequence of light chain.

An embodiment provides a kind of antibody or its function equivalent, and it comprises the sequence of heavy chain be made up of sequence of heavy chain shown in Figure 14 B and the sequence of light chain be made up of sequence of light chain shown in Figure 14 B.Or, as well known to the skilled person, may produce and shorten but maintain heavy chain or the sequence of light chain of interested binding characteristic.Preferably, compared with initial heavy chain or light chain, heavy chain or the light chain of this shortening of generation have shorter constant region.Preferred maintenance variable region is constant.Such as, Fab fragment or F (ab') is produced based on sequence of heavy chain shown in Figure 14 B or sequence of light chain ₂fragment.Therefore, the antibody function equivalent of the Functional portions at least comprising sequence shown in Figure 14 B is also provided.The length of described Functional portions is at least 20 amino acid, comprises the sequence identical with at least one CDR sequence at least 70% shown in Figure 14 B.

The particularly preferred anti-RSV antibodies of another kind of the present invention is the antibody being called " AM23 ", and it has heavy chain district as shown in Figure 14 C and light chain district.The CDR sequence of the AM23 of the antigenic binding property of concrete generation AM23 is also see Figure 14 C.

Since the present invention recognizes that CDR sequence shown in Figure 14 C provides required RSV binding characteristic, technician just can produce the variant comprising the CDR sequence that at least one changes.Such as, apply conservative amino acid to replace.Conservative amino acid replaces another kind of aminoacid replacement one seed amino acid comprised with character (size, hydrophobicity etc.) basic simlarity, such that general function is unlikely to be had a strong impact on.

Also at least one CDR sequence shown in Figure 14 C may be changed, to produce antibody variants or its function equivalent of at least one characteristic changing compared with AM23.Preferably, the antibody provided or function equivalent comprise the CDR sequence identical with CDR sequence at least 70% shown in Figure 14 C, to keep, even to improve the favourable binding characteristic of AM23 at least partly.CDR sequence shown in preferred change Figure 14 C, the characteristic comprising at least one to make the antibody that obtains or function equivalent compared with AM23 and improve, the binding affinity such as improved, selectivity and/or stability.Therefore, the antibody variants or its function equivalent that comprise the aminoacid sequence identical with CDR sequence at least 70% shown in Figure 14 C belong to the scope of the invention.This area can obtain the method for various change aminoacid sequence.Such as, synthetic has heavy chain or the sequence of light chain of required CDR sequence.Preferably, utilize (such as) random or site-directed mutagenesis that the nucleotide sequence of coding CDR is suddenlyd change.

-heavy chain CDR1 sequence, it comprises the sequence identical with sequence GFNFHNYG at least 70%, and/or

-heavy chain CDR2 sequence, it comprises the sequence identical with sequence VWYDGSKK at least 70%, and/or

-heavy chain CDR3 sequence, it comprises the sequence identical with sequence VRDKVGPTPYFDS at least 70%, and/or

-light chain CDR1 sequence, it comprises the sequence identical with sequence NIGSET at least 70%, and/or

-light chain CDR2 sequence, it comprises the sequence identical with sequence D DD at least 70%, and/or

-light chain CDR3 sequence, it comprises the sequence identical with sequence QVWDRSNYHQV at least 70%.

Preferably, antibody of the present invention or function equivalent comprise and at least one CDR sequence at least 75% shown in Figure 14 C, and more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical CDR sequence.Most preferably, antibody of the present invention or function equivalent comprise the CDR sequence identical with at least one CDR sequence at least 95% shown in Figure 14 C.Above-mentioned particularly preferred antibody A M23 comprises the CDR sequence be made up of CDR sequence shown in Figure 14 C.Therefore, the particularly preferred embodiment of the present invention provide a kind of separation, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its function equivalent, it comprises:

-heavy chain CDR1 sequence, it comprises sequence GFNFHNYG, and/or

-heavy chain CDR2 sequence, it comprises sequence VWYDGSKK, and/or

-heavy chain CDR3 sequence, it comprises sequence VRDKVGPTPYFDS, and/or

-light chain CDR1 sequence, it comprises sequence NIGSET, and/or

-light chain CDR2 sequence, it comprises sequence D DD, and/or

-light chain CDR3 sequence, it comprises sequence QVWDRSNYHQV.

In one embodiment, the antibody provided or function equivalent comprise three heavy CDR sequences as shown in Figure 14 C and three CDR sequence, or with its at least 70% identical sequence.Therefore, there is provided a kind of to be further separated, the antibody of synthesis or restructuring or its function equivalent, it comprises the heavy chain CDR1 sequence containing the sequence identical with sequence GFNFHNYG at least 70%, heavy chain CDR2 sequence containing the sequence identical with sequence VWYDGSKK at least 70%, heavy chain CDR3 sequence containing the sequence identical with sequence VRDKVGPTPYFDS at least 70%, light chain CDR1 sequence containing the sequence identical with sequence NIGSET at least 70%, light chain CDR2 sequence containing the sequence identical with sequence D DD at least 70% and the light chain CDR3 sequence containing the sequence identical with sequence QVWDRSNYHQV at least 70%.Described antibody or function equivalent preferably comprise with the above-mentioned heavy CDR sequences shown in Figure 14 C and above-mentioned CDR sequence at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical CDR sequence.Also providing package contains antibody or the function equivalent of heavy chain CDR1, CDR2 and CDR3 sequence of above-mentioned Figure 14 C and light chain CDR1, CDR2 and CDR3 sequence of above-mentioned Figure 14 C.

Also providing package contains antibody or its function equivalent of the heavy chain amino acid sequence identical with sequence of heavy chain at least 70% shown in Figure 14 C.This kind of sequence of heavy chain provides required RSV binding characteristic, as antibody A M23 prove.Therefore, also provide a kind of antibody or its function equivalent, its sequence of heavy chain comprises the sequence identical with sequence EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWVRQAPGKGLEWVAVVWYDGS KKYYADSVTGRFAISRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVGPTPYFDSWGQ GTLVTVSS at least 70%.And the light-chain amino acid sequence identical with sequence of light chain at least 70% shown in Figure 14 C provides required RSV binding characteristic, as antibody A M23 prove.Therefore, also provide a kind of antibody or its function equivalent, it has the sequence of light chain identical with sequence SYVLTQPPSVSLAPGGTAAITCGRNNIGSETVHWYQQKPGQAPVLVVYDDDDRPSG IPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSNYHQVFGGGTKLTV at least 70%.Antibody of the present invention or Functional portions preferably comprise with sequence of heavy chain shown in Figure 14 C and/or sequence of light chain at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical weight chain variabl area sequence and/or light-chain variable sequence.Homology is higher, and described antibody or Functional portions are more close to antibody A M23.Antibody of the present invention or Functional portions preferably comprise the heavy chain of similar AM23 and the heavy chain of light chain and light chain.Therefore, a kind of antibody or Functional portions are also provided, its comprise with sequence of heavy chain shown in Figure 14 C and sequence of light chain at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical sequence of heavy chain and sequence of light chain.

An embodiment provides a kind of antibody or its function equivalent, and it comprises the sequence of heavy chain be made up of sequence of heavy chain shown in Figure 14 C and the sequence of light chain be made up of sequence of light chain shown in Figure 14 C.Or, as well known to the skilled person, may produce and shorten but maintain heavy chain or the sequence of light chain of binding characteristic interested.Preferably, compared with initial heavy chain or light chain, heavy chain or the light chain of this shortening of generation have shorter constant region.Preferred maintenance variable region is constant.Such as, Fab fragment or F (ab') is produced based on sequence of heavy chain shown in Figure 14 C or sequence of light chain ₂fragment.Therefore, the antibody function equivalent of the Functional portions at least comprising sequence shown in Figure 14 C is also provided.The length of described Functional portions is at least 20 amino acid, comprises the sequence identical with at least one CDR sequence at least 70% shown in Figure 14 C.

The invention provides the RSV-specific antibody or its function equivalent that to have compared with prior art antibody and improve characteristic.Present inventor successfully produces the RSV specific antibody with low IC50 value.This antibody-like is high especially or strong especially to the avidity of RSV, is therefore particularly suitable for the detrimentally affect resisting and/or prevent at least partly rsv infection and/or rsv infection.An embodiment is provided in IC in the external Neutralizing test of HEp-2 cell infection RSV ₅₀value is less than the antibody of 10ng/ml and the function equivalent of described antibody.The IC of described antibody or function equivalent ₅₀value is preferably less than 5ng/ml, is more preferably less than 2ng/ml.In the external Neutralizing test described in embodiment, the IC of preferred antibody D25 ₅₀value is about 0.5-1.5ng/ml (see Fig. 8).

Antibody of the present invention is preferably people's antibody.People's antibody is used for the treatment of people can reduce because individual human to produce the probability of the side effect caused by immune response to nonhuman sequence.In another preferred embodiment, antibody of the present invention or Functional portions, derivative or analogue are chimeric antibodies.In this way, can by interested sequence, such as interested binding site is included in antibody of the present invention or function equivalent.

The present invention also provides separation, the code book invention antibody of synthesis or restructuring or the nucleotide sequence of function equivalent or its Functional portions, derivative or analogue.Such as, this kind of separate nucleic acid is from the B cell that can produce antibody of the present invention, as described in greater detail below.Preferred embodiment providing package containing at least with the nucleotide sequence of the sequence of Functional portions at least 70% homology of nucleotide sequence shown in Figure 11, Figure 12, Figure 14 A, Figure 14 B and/or Figure 14 B.Described nucleotide sequence preferably comprise at least with the sequence of Functional portions at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% homology of nucleotide sequence shown in Figure 11, Figure 12, Figure 14 A, Figure 14 B and/or Figure 14 B.The length of described Functional portions is at least 30 Nucleotide, preferably at least 50 Nucleotide, more preferably at least 75 Nucleotide.Preferably, at least one nucleotide sequence shown in described Functional portions code pattern 11D, Figure 12, Figure 14 A, Figure 14 B and/or Figure 14 B.Described sequence preference is CDR sequence.

Antibody of the present invention or function equivalent are especially suitable for use as medicine or preventive.Antibody of the present invention as medicine and/or preventive or its Functional portions, derivative or analogue are also provided herein.In particularly preferred embodiments, described antibody comprises antibody D25, AM14, AM16 and/or AM23, or its Functional portions, derivative or analogue.Described medicine or preventive are preferred for opposing or prevent rsv infection at least partly, or the detrimentally affect of opposing or at least part of prevention rsv infection.Therefore, also provide antibody of the present invention, Functional portions, derivative or analogue for the preparation of the application treated and/or prevented at least partly in the medicine of RSV relative disease and/or preventive, and for treating and/or preventing the method for RSV relative disease at least partly, described method comprises antibody of the present invention or the function equivalent of the individual treatment significant quantity giving needs.Described antibody preferably includes antibody D25, AM14, AM16 and/or AM23, or its Functional portions, derivative or analogue.

In order to resist RSV, antibody of the present invention or function equivalent preferably gave individuality before generation rsv infection.Or, give antibody of the present invention or function equivalent when individuality infects RSV.Described antibody or function equivalent preferably give the individuality of suffering from the raising of RSV relative disease risk, as premature infant, suffer from chronic lung disease, congenital heart disease and/or immunoincompetent individuality, and the children below 6 week age.The risk that the elderly suffers from RSV relative disease also raises.Antibody of the present invention or function equivalent give preferably by oral or one or many injection.Clinical dosage in the clinical trial of strictly developing programs as required increases progressively research, is designed for the antibody of the present invention for the treatment of use described herein and/or the dosage range of function equivalent.Typical doses is 0.1 to 10mg/kg body weight.In treatment use, antibody of the present invention or function equivalent are general with pharmaceutically acceptable carrier, auxiliary material, thinner and/or vehicle conbined usage.Such as, the example of suitable carrier comprises keyhole keyhole limpet hemocyanin (KLH), serum albumin (as BSA or RSA) and ovalbumin.Those skilled in the art understand many suitable oil bases and water base auxiliary material.In one embodiment, described auxiliary material comprises Specol.In another embodiment, described suitable carrier comprises solution, as salt solution.

In another embodiment, the nucleic acid of code book invention antibody or Functional portions is used.After giving this nucleic acid, produce antibody or function equivalent by the cellular machineries of host.The antibody produced or function equivalent can prevent and/or resist the detrimentally affect of rsv infection and/or rsv infection.Therefore, also provide herein as the nucleotide sequence of the present invention of medicine and/or preventive, Functional portions, derivative and/or analogue.Described nucleic acid is preferred for resisting RSV.Therefore, nucleotide sequence of the present invention, Functional portions, derivative and/or analogue is also provided to prepare the application in the medicine and/or preventive treating and/or preventing RSV relative disease at least partly.

The Functional portions being at least nucleic acid of the present invention refers to a part for described nucleic acid, length is at least 30 base pairs, preferred at least 50 base pairs, more preferably at least 100 base pairs, it comprises at least one expression characteristic (kind identical, and amount not necessarily identical) identical with nucleic acid of the present invention.Described Functional portions at least encoded packets contains the aminoacid sequence of the sequence identical with CDR sequence at least 70% shown in Figure 11 D, Figure 14 A, Figure 14 B and/or Figure 14 C.

The present invention also provides the cell producing separation antibody, and it can produce antibody of the present invention, Functional portions, derivative or analogue.Possibility (and non-limiting) mode of the cell obtaining this kind of generation antibody is described in detail in embodiment.The present inventor's exploitation and use novel method improve the stability of the cell producing RSV specific antibody.Make the RSV specific antibody produced in this way produce cell and at least stablize 6 months.Therefore, the present invention also provides the RSV specific antibody generation cell of the present invention stablizing at least 9 weeks, preferably at least 3 months, more preferably at least 6 months.

Present inventor recognizes, producing the amount of BCL6 and/or Blimp-1 expression product in cell, can realize the stability of described antibody produced cell by affecting RSV specific antibody.The amount of BCL6 and/or Blimp-1 expression product is directly or indirectly affected.Preferably, the amount of BCL6 and Blimp-1 expression product in described antibody produced cell is all made adjustments, because these two kinds of expression products all relate to the stability of antibody produced cell.The definition of stability of antibody produced cell is the ability (preferably after described cell enters the described stage) that described antibody produced cell remains on certain etap.The different developmental phases of cell comprises the feature of the different described cell of at least one.Such as, known memory B cell is referred to as the stage differentiated one-tenth antibody-secreting type plasmocyte of plasmablast after stimulation by some investigators.Memory B cell, plasmablast and plasmocyte are that wherein B cell has the different developmental phases of the B cell of different characteristics.Memory B cell has low propagation and low antibody-secreting characteristic.Plasmablast has the gentle higher antibody-secreting level of higher proliferation water compared with memory B cell, and plasma cell secretion High antibody level, but can not breed.What the method described in present inventor of use may regulate antibody produced cell copies the life-span.In this article, the life-span of copying of antibody produced cell is defined as B cell and its progeny cell and can copies and maintain its time of ability producing antibody and/or develop into antibody produced cell.Preferred prolongation antibody produced cell copy the life-span, this means that end differentiation eventually will not occur described antibody produced cell, or only just occurs after extended periods lastly to break up end compared with the antibody produced cell of the identical type used at present, and continuous proliferation in vitro.According to the present invention, the amount of BCL6 and/or Blimp-1 expression product in antibody produced cell may be regulated, make this antibody produced cell enter and/or remain on the developmental condition of predetermined cell continuous proliferation.Therefore, what use the inventive method may extend antibody produced cell copies the life-span, because likely B cell to be maintained certain etap of copying.See the PCT/NL2006/000625 that the applicant submits to.The invention provides and produce the stable celliferous mode of RSV specific antibody and method.

Antibody produced cell is defined as and can produces and/or the cell of secretory antibody or its function equivalent, and/or can develop into and can produce and/or the cell of cell of secretory antibody or its function equivalent.In this article, RSV specific antibody produces cell and is defined as cell and can produces and/or secretory antibody or its function equivalent, described antibody or its function equivalent can specific binding RSV and/or RSV components, as the epi-position of RSV F (fusion) albumen, RSV G (attached) albumen or RSV SH (little hydrophobic) albumen.Preferably, described RSV specific antibody produces cell and comprises B cell and/or the derivative plasmocyte of B cell.B cell is referred to herein as antibody produced cell, even be in antibody production in this B cell very low or do not produce stage of antibody, as activation or not activated origi-nal B-cell or memory B cell, because this cell can develop into the cell producing antibody, as plasmablast and/or plasmocyte.

RSV specific antibody of the present invention produces cell and preferably comprises mammalian cell.Non-limitative example comprises the antibody produced cell of derived from human individuality, rodent, rabbit, camel, pig, ox, goat, horse, ape, gorilla.Described antibody produced cell preferably includes people's cell, mouse cell, rabbit cell and/or camel cell.

BCL6 encode normal B cells and T cell growth and maturation is required and germinal center forms required transcription repressor.(Ye，1997)。BCL6 is high expression level in Germinal center B cell, and expresses hardly in plasmocyte.BCL6 suppresses activating B cell to plasmacytic differentiation.The maturation protein-1 (Blimp-1) of transcription repressor bone-marrow-derived lymphocyte induction is that B cell develops into the required factor of plasmocyte.People's variant of Blimp-1 is called Prdm1.As used herein, the place of any Blimp-1 of mentioning includes Prdm1.Blimp-1 drives plasma cell differentiation.BCL6 and Blimp-1 suppresses mutually to express; Therefore, under natural conditions, when wherein a kind of expression level reached is higher than another kind, force to reach certain differentiation state.In human body, from activation origi-nal B-cell or memory B cell be divided into plasmocyte relate to BCL6 lower mediation Blimp-1 rise.In germinal center, cell BCL6 expression level is high, and Blimp-1 expression level is low.In the memory cell of tranquillization, the expression level of BCL6 and Blimp-1 is all lower.The signal triggering differentiation causes Blimp-1 to raise, and this kind of Blimp-1 resists the expression of BCL6.The stage that BCL6 and Blimp-1 all expresses is very short, is called plasmablast.Along with improving constantly of Blimp-1 level, BCL6 expresses disappearance, causes forming plasmocyte.

In an embodiment of the invention, the RSV specific antibody of BCL6 and Blimp-1 co expression is provided to produce cell (meaning that BCL6 and Blimp-1 all expresses at least 1 day, preferred at least 1 week, more preferably at least 6 weeks, most preferably at least 3 months in described antibody produced cell).When providing suitable signal, described RSV specific antibody produces cell and can breed.Find, BCL6 and Blimp-1 co expression produces the antibody produced cell can breeding and produce antibody.BCL6 and Blimp-1 preferably in B cell, co expression in preferred human B cell.BCL6 and Blimp-1 co expression in B cell causes described B cell to be stabilized in the plasmablast shape stage.Plasmablast is similar to plasmocyte, can secretory antibody.But plasmablast still can be bred, and plasmocyte loses multiplication capacity.Therefore, plasmocyte is not suitable for cultivating antibody produced cell system.

One preferred embodiment provides RSV specific antibody to produce cell, and it comprises the exogenous nucleic acid sequence of coding BCL6 or its Functional portions, derivative and/or analogue.Exogenous Nucleic Acid is defined as the nucleotide sequence not belonging to cellular genome under its natural environment in this article.Use this kind of exogenous nucleic acid molecules, the BCL6 concentration in antibody produced cell may be regulated when not relying on endogenous BCL6 and expressing.Therefore, even if the expression level of endogenous BCL6 is lower or do not express (such as Blimp-1 is caused), the exogenous nucleic acid sequence of coding BCL6 or its Functional portions, derivative and/or analogue still can produce the BCL6 of the concentration enough affecting antibody produced cell stability.The nucleotide sequence of described coding BCL6 or its Functional portions, derivative and/or analogue is preferably as constitutive activity, is expressed by endogenous repressor even if therefore express at the endogenous BCL6 of described cell as also maintained BCL6 when Blimp-1 suppresses.Most preferably, the expression of the nucleotide sequence of described coding BCL6 or its Functional portions, derivative and/or analogue, by the adjustment of the exogenous inducer of repressor, makes it possible to the degree regulating arbitrarily BCL6 to express.

Preferably, as detailed below, RSV specific antibody of the present invention produces the exogenous nucleic acid sequence that cell comprises coding Bcl-xL or its Functional portions, derivative and/or analogue.If there is Bcl-xL or its Functional portions, derivative and/or analogue, then may cultivate plasmablast under the condition of low cell density.Preferably, the expression of the nucleotide sequence of described coding Bcl-xL or its Functional portions, derivative and/or analogue, by the adjustment of the exogenous inducer of repressor, makes it possible to the degree regulating arbitrarily Bcl-xL to express.Therefore, a preferred implementation provides a kind of RSV specific antibody to produce cell, and it comprises:

The exogenous nucleic acid sequence of-coding BCL6 or its Functional portions, derivative and/or analogue, and/or

The exogenous nucleic acid sequence of-coding Bcl-xL or its Functional portions, derivative and/or analogue.

Described RSV specific antibody produces cell and preferably comprises the exogenous nucleic acid sequence of coding BCL6 or its Functional portions, derivative and/or analogue and the exogenous nucleic acid sequence of coding Bcl-xL or its Functional portions, derivative and/or analogue simultaneously.Preferably, the activator of expression by exogenous compounds induction of the nucleotide sequence of described coding BCL6, Bcl-xL or BCL6 or the Functional portions of Bcl-xL, derivative and/or analogue and/or the adjustment of repressor.Such as, inducible promoter systems, such as Tet-on or Tet-off system is used.

Stable RSV specific antibody of the present invention generation cell is produced preferably by producing co expression BCL6 and Blimp-1 in cell at RSV specific antibody.RSV specific antibody produces cell preferably available from the individuality contacting RSV.The celliferous method of separation antibody is known in the art.Such as, the RSV derivative compound of marking with marker or label is hatched together with the sample of the individuality contacting RSV, and this sample comprises antibody produced cell.The RSV specific antibody being separated the RSV derivative compound identifying this mark produces cell, washes unconjugated cell off simultaneously.Subsequently, the RSV specific antibody obtained is made to produce cell stabilization by co expression BCL6 and Blimp-1.

An embodiment comprises, and the first stable whole antibody produced cells contacting donor from RSV, are then separated the cell of the RSV derivative compound of identification marking.In another embodiment, antibody produced cell is at its B-cell receptor (BCR, the film expression form of antibody) downstream has (fluorescence) marker, described acceptor at antibody produced cell by conducted signal during the antigen of BCR in conjunction with unmarked/non-label.Select the antibody produced cell that this marker overturns, make it stablize by co expression BCL6 and Blimp-1 subsequently.In another embodiment, do not having antigen derivative compound to use, but when having experiment to can be used for screening unique antibodies, by co expression BCL6 and Blimp-1, optionally also expressing Bcl-XL and stablize all/large quantities of antibody produced cell.According to this embodiment, with low density cell culture cell under L cell exists, preferably 10 to 100 cells (short run culture, MBC) in the 96 every holes of orifice plate.Culture supernatants can be directly used in screening experiment, and such as ELISA, western blot or function test are as ELISPOT, Neutralizing test or Cell migration assay.

In one embodiment, select MBC, in order to obtain the monoclonal cell system of antibody produced cell interested, limiting dilution being carried out to culture, preferably after 2-3 week, again screening the supernatant liquor of these cultures with optimization experiment.

As well known to the skilled person, this area can obtain many replacement methods.Above-mentioned embodiment is nonrestrictive.

Therefore, also provide a kind of method producing antibody produced cell, it is stablized at least three months and can produce RSV-specific antibody or its function equivalent, and described method comprises:

-improve the Blimp-1 expression level that can produce in the cell of RSV-specific antibody or its function equivalent; With

-improve and/or maintain the BCL6 expression level in described cell.

Utilize the inventive method, may RSV specific memory B-cell be changed into plasmablast like cell, and stablize described cell, therefore can not be divided into plasmocyte fast.These are different from plasmacytic natural growth, and wherein the expression of Blimp-1 in memory B cell causes Rapid development plasmablast, thus suppress BCL6 to express, and the plasmocyte therefore obtained expresses BCL6 hardly.Therefore, an embodiment of the invention are included in co expression BCL6 and Blimp-1 in RSV specific b cells, produce the cell can breeding and produce antibody.In described RSV specific b cells, BCL6 expression level is preferably adjusted to or maintains the level identical or higher with plasmablast.Produce the stable culture of RSV specific b cells in this way, this cell keeps the ability producing RSV-specific antibody.Preferably by adding anti-apoptogene Bcl-xL, stablize these RSV specific b cells of co expression BCL6 and Blimp-1 further.By introducing Bcl-xL, plasmablast may be cultivated under low cell density condition now.Therefore, the present invention also provides a kind of method of cultivating plasmablast under the condition of low cell density, and the RSV specific antibody that described method comprises with any method generation as herein described has BCL6, Blimp-1 and Bcl-xL expression level produces cell.

BCL6 expression product (preferred BCL6 albumen) is regulated to produce the content in cell at RSV specific antibody by various mode.

In one embodiment, the compound that directly or indirectly can affect BCL6 and express is provided to antibody produced cell.The compound that can strengthen BCL6 and express preferably is provided to antibody produced cell, lowers with the BCL6 resisted during Blimp-1 expresses.This compounds preferably comprises signal transduction and Activator protein 5 (STAT5) or its Functional portions, derivative and/or analogue, and/or their nucleotide sequence of encoding.STAT5 is the signal transducer that can strengthen BCL6 expression.STAT5 has two kinds of form known STAT5a and STAT5b, and they are encoded by two kinds of different tandem genes.Giving and/or activate STAT5 causes BCL6 level to improve.Therefore, STAT5 or its Functional portions, derivative and/or analogue compensate for the downward effect of Blimp-1 to BCL6 at least partly to the rise effect that BCL6 expresses.Therefore, STAT5 or its Functional portions, derivative and/or analogue directly can affect BCL6 expression.Also may express by remote effect BCL6.Such as, this realizes by regulating the consumption of the compound that directly or indirectly can activate STAT5 and/or regulate STAT5 to express.Therefore, in one embodiment, expression and/or the activity of endogenous and/or exogenous STAT5 is improved.Such as, antibody produced cell can be cultivated under existing at the interleukin (IL) 2 and/or IL4 that can activate STAT5, indirectly strengthen BCL6 and express.

In one embodiment, the nucleotide sequence that cell provides coding STAT5 or its Functional portions, derivative and/or analogue is produced to RSV specific antibody, wherein said nucleotide sequence has constitutive activity, the existence of the instrumentality that this means that STAT5 does not rely on (endogenous) and continuous expression.Lower or when not expressing at endogenous STAT5 expression level, the exogenous constitutive activity nucleotide sequence preferably applying coding STAT5 or its Functional portions, derivative and/or analogue causes the concentration of STAT5 or its Functional portions, derivative and/or analogue to be enough to increase BCL6 expressing.Most preferably, producing cell to RSV specific antibody provides encoded packets to contain the compound of STAT5 or its Functional portions, derivative and/or analogue, the nucleotide sequence of preferred fusion rotein, the adjustment of its active exogenous inducer by repressor, the activation degree that therefore can arbitrarily regulate BCL6 to express.There is provided another system Tet-on system of induction BCL-6, wherein add the activity of tsiklomitsin and/or tetracycline derivant induced transactivation albumen, trans-activator can induce transcribing of BCL6 gene, is then the synthesis of BCL albumen.In a preferred embodiment, the nucleotide sequence of the fusion protein ER-STAT5 of coding estrogen receptor (ER) and STAT5 is provided to antibody produced cell.This kind of fusion rotein non-activity, because it forms mixture with heat shock protein in endochylema.STAT5 can not arrive nucleus in this way, cannot improve BCL6 and express.After giving exogenous inducer 4 hydroxyls-tamoxifen (4HT), fusion protein ER-STAT5 and heat shock protein dissociate, and therefore STAT5 can enter nucleus and activate BCL6 and express.

In addition or or, cultivate described antibody produced cell can directly or indirectly strengthen under compound that BCL6 expresses exists, thus improve the BCL6 that RSV specific antibody produces in cell and express.

Therefore, a kind of embodiment provides a kind of and produces the celliferous method of RSV specific antibody, and described method comprises:

-produce cell to RSV specific antibody to provide the compound that directly or indirectly can strengthen BCL6 and express; And/or

-under the compound that directly or indirectly can strengthen BCL6 expression exists, cultivate RSV specific antibody produce cell.

The described compound that directly or indirectly can strengthen BCL6 expression preferably comprises STAT5 or its Functional portions, derivative and/or analogue.Therefore, the invention provides a kind of method, it comprises provides STAT5 or its Functional portions, derivative and/or analogue to described RSV specific antibody generation cell, or the nucleotide sequence of coding STAT5 or its Functional portions, derivative and/or analogue.In one embodiment, after the nucleotide sequence of coding STAT5 or its Functional portions, derivative and/or analogue is introduced described cell, described antibody produced cell is cultivated.Such as, by transfection and/or virus-mediated transgenosis, described nucleotide sequence is introduced described cell.This area can obtain many alternative methods nucleotide sequence being introduced cell, here need not explain further.

Use directly or indirectly can strengthen the compound that BCL6 expresses, and may improve the expression of endogenous BCL6.But, in one preferred embodiment, provide the nucleotide sequence of coding BCL6 or its Functional portions, derivative and/or analogue to antibody produced cell.As previously mentioned, the Exogenous Nucleic Acid of optimized encoding BCL6, because this can lower the BCL6 concentration in ganglion cell in the situation not relying on endogenous BCL6 expression.Therefore, even if the expression level of endogenous BCL6 is lower or do not express (such as Blimp-1 is caused), the exogenous nucleic acid sequence of coding BCL6 or its Functional portions, derivative and/or analogue still can produce the BCL6 of the concentration enough affecting antibody produced cell stability.Therefore, the present invention also provides a kind of method, and it comprises the nucleotide sequence providing coding BCL6 or its Functional portions, derivative and/or analogue to RSV specific antibody generation cell.Preferably, the constitutive activity nucleotide sequence of coding BCL6 or its Functional portions, derivative and/or analogue is provided to described antibody produced cell, thus when the endogenous BCL6 expression of described cell is suppressed as Blimp-1 by endogenous repressor, maintain BCL6 and express.Most preferably, regulated the expression of the described nucleotide sequence of coding BCL6 or its Functional portions, derivative and/or analogue by the exogenous inducer of repressor, thus arbitrarily regulate the expression degree of BCL6.Such as, inducible promoter systems on the books is used, such as Tet-on or Tet-off system.

Another preferred embodiment in, the invention provides a kind of method, wherein providing coding E47 or the nucleotide sequence indirect regulation BCL6 content of its Functional portions, derivative and/or analogue by producing cell to RSV specific antibody.The transcription factor of E47 coding belongs to helix-loop-helix protein matter family, i.e. E-protein family.Have four kinds of E-albumen, namely E12, E47, E2-2 and HEB participate in lymphocyte development.E12 and E47 is encoded by a kind of gene and E2A, but montage is different.E-albumen can by E protein inhibitor Id2 and Id3, and ABF-1 suppresses (Mathas S., 2006).E protein is described to tumor inhibitor, and its overexpression can be apoptosis-induced.One of specificity target spot of E47 is Socs1 and Socs3 gene.These Socs genes are called as the negative regulator gene of STAT5b, because of but the indirect regulation agent of BCL6.In other words, the expression of E47 in B cell enhances Blimp-1 and expresses, and this causes B cell to produce phenotype (plasmocyte) differentiation to antibody.

Also Blimp-1 is regulated to produce the expression amount in cell at RSV specific antibody by various mode.In one embodiment, produce cell to RSV specific antibody and the compound that directly or indirectly can affect Blimp-1 and express is provided.In addition or or, can directly or indirectly affect Blimp-1 express compound exist under cultivate antibody produced cell.Therefore, the present invention also provides a kind of method, and it comprises provides the compound that directly or indirectly can affect Blimp-1 and express to RSV specific antibody generation cell.The present invention also provides a kind of method, and it is included in the described antibody produced cell of the lower cultivation of compound existence that directly or indirectly can affect Blimp-1 and express.Preferred use can strengthen the compound that Blimp-1 expresses, to resist the downward that BCL6 expresses period Blimp-1.Described compound most preferably comprises IL-21.

In a preferred embodiment, directly or indirectly can affect the described compound that Blimp-1 expresses and comprise signal transduction and Activator protein 3 (STAT3) albumen or its Functional portions, derivative and/or analogue, and/or their nucleotide sequence of encoding.STAT3 is the signal transducer participating in B cell growth and differentiation.STAT3 can raise Blimp-1 and express.Therefore, the present invention also provides a kind of method, the described compound that wherein directly or indirectly can affect Blimp-1 expression comprises STAT3 or its Functional portions, derivative and/or analogue, or the nucleotide sequence of coding STAT3 or its Functional portions, derivative and/or analogue.Most preferably, regulated the expression of the described nucleotide sequence of coding STAT3 or its Functional portions, derivative and/or analogue by the exogenous inducer of repressor, thus arbitrarily regulate the expression degree of STAT3.Such as, inducible promoter systems, such as Tet-on or Tet-off system is used.In one embodiment, the fusion product comprising STAT3, derivative or analogue and ER is introduced described cell, to regulate STAT3 to express by trans-Hydroxytamoxifen.

Express because STAT3 can affect Blimp-1, also can by giving directly or indirectly to regulate the activity of STAT3 and/or the compound indirect regulation Blimp-1 of expression to express.In one embodiment, provide the compound that can strengthen STAT3 activity to antibody produced cell, indirectly to improve the expression of Blimp-1.Therefore, the present invention also provides a kind of method, wherein provides the compound that directly or indirectly can strengthen STAT3 activity to antibody produced cell.

Therefore, in one embodiment, provide the compound that directly or indirectly can activate STAT3 to antibody produced cell, express to improve Blimp-1.

Activate STAT3 in every way.Preferably, by providing cytokine activation STAT3 to antibody produced cell.The cytokine participating in B cell differentiation under its natural environment effectively can regulate stat protein.The very effective activator of STAT3 is IL-21 and IL-6, and known IL-2, IL-7, IL-10, IL-15 and IL-27 also can activate STAT3.And the Toll-sample acceptor (TLR) participating in congenital immunity also can activate STAT3.Therefore, an embodiment of the invention provide a kind of method, and the described compound that wherein directly or indirectly can affect Blimp-1 expression comprises IL-21, IL-2, IL-6, IL-7, IL-10, IL-15 and/or IL-27.Most preferably use IL-21, because IL-21 is particularly suitable for the stability affecting antibody produced cell.Even if when BCL6 resists Blimp-1 expression, IL-21 also can raise Blimp-1 and express.

In addition or or, use sudden change Zhan Nasi kinases (JAK) to activate STAT3.Under natural condition, JAK self is by can pSTAT3 after at least one cytokine activation.The existence of cytokine can not be relied on and the Zhan Nasi kinases activating the sudden change of STAT3 is specially adapted to the inventive method.

As previously mentioned, the compound that can strengthen Blimp-1 expression in one embodiment comprises the nucleotide sequence of coding STAT3 or its Functional portions, derivative and/or analogue.Even if exist coding STAT3 or the exogenous nucleic acid sequence of its Functional portions, derivative and/or analogue make very low in the expression of endogenous STAT3 or do not express time, still sustainable existence STAT3 or its Functional portions, derivative and/or analogue.

Also expression and/or the activity of STAT5 may be reduced, to raise Blimp-1.If the content of STAT5 and/or active reduction, also reduce the activation that BCL6 expresses, this causes BCL6 expression product content to reduce.Because BCL6 and Blimp-1 resists expression mutually, Blimp-1 expression product amount is caused to increase so BCL6 expression product amount reduces.Therefore, it is possible to the compound lowering STAT5 activity can raise Blimp-1 indirectly.Such as, this compounds comprises cytokine signaling conduction arrestin (SOCS).Therefore, in one embodiment, by providing SOCS albumen to described cell, and/or activating SOCS albumen in described cell, the content that RSV specific antibody produces Blimp-1 expression product in cell can be raised.

In a preferred embodiment, when providing the nucleotide sequence of coding E47 or its Functional portions, derivative and/or analogue to RSV-specific antibody generation cell, the expression of STAT5 and/or active reduction.In the B cell of high level expression STAT5b, express E47 interfered Differentiation and proliferation, namely block STAT5 by E47 and SOCS and cause BCL6 level to reduce, Blimp-1 level rises subsequently.Blimp-1 level raises and causes propagation level to decline, and causes involved cell to the differentiation of antibody produced cell.In other words, in B cell, express E47 strengthen Blimp-1 expression, this causes B cell to produce phenotype (plasmocyte) differentiation to antibody.

At least the Functional portions of STAT5 albumen, STAT3 albumen, Bcl-xL and/or BCL6 refers to compared with STAT5 albumen, STAT3 albumen, Bcl-xL and/or BCL6, and the ability affecting the stability of antibody produced cell is identical with – kind, amount differs the proteinaceous molecule of phasing with –.Such as, the Functional portions of STAT5 albumen or STAT3 albumen is not containing the amino acid not participating in or seldom participate in described ability.The derivative of STAT5 albumen, STAT3 albumen, Bcl-xL and/or BCL6 is defined as through changing, and the capability class of the stability of described protein influence antibody produced cell is substantially identical and measure not necessarily identical protein.In many ways, such as replaced by conservative amino acid and provide derivative, one of them amino acid by another aminoacid replacement of character (size, hydrophobicity etc.) basic simlarity, such that general function is unlikely to be had a strong impact on.Such as, derivative comprises fusion rotein, and as STAT5-ER or STAT3-ER fusion rotein, its activity depends on the existence of 4 hydroxyls-tamoxifen (4HT).The analogue of STAT5 albumen, STAT3 albumen, Bcl-xL and/or BCL6 is defined as the identical and molecule that amount is not necessarily identical of the capability class affecting antibody produced cell stability.Described analogue is not necessarily derived from described STAT5 albumen, STAT3 albumen, Bcl-xL and/or BCL6.

In a preferred embodiment, before the nucleotide sequence that coding BCL6 or its Functional portions, derivative and/or analogue are provided to described antibody produced cell, under IL-21 exists, cultivate described RSV specific antibody produce cell.Preferably before the nucleotide sequence that coding BCL6 or its Functional portions, derivative and/or analogue are provided to described cell, under IL-21 exists, cultivate RSV specific antibody produce cell, preferred B cell, because in these embodiments, stability, propagation and/or antibody produce and are improved especially.

In a preferred embodiment, the invention provides a kind of method affecting the celliferous stability of RSV specific antibody described herein, the method also comprises the content directly or indirectly improving Bcl-xL expression product in described antibody produced cell.Such as, by providing the nucleotide sequence of coding Bcl-xL or its Functional portions, derivative and/or analogue or other anti-apoptotic genes expressions of encoding to described antibody produced cell, include but not limited to that the nucleotide sequence of Bcl-2 realizes above-mentioned purpose.In another embodiment, by providing the compound that directly or indirectly can improve Bcl-xL expression to realize this purpose to described antibody produced cell, described compound comprises APRIL, BAFF, CD40, BCR stimulation, cytokine, somatomedin or downstream effect thing sample JNK and AKT (PKB).

Bcl-xL is anti-apoptotic Bcl-2 family member, Bcl2 albumen and the so-called family member only containing Bcl-2 homeodomain 3 (BH3) interact as Bax, Bak, Bim and Bad or resist, after receiving intrinsic death stimulation, induced cytochrome c discharges (Boise, L.H., 1993).Therefore, most important for cell survival by the integrity of the protein protection mitochondrial membranes such as such as Bcl-xL.

Prove, STAT5 intensity of activation Cell protection avoids necrocytosis occurs.STAT5 can regulate the expression of Bcl-xL, and this supports the Anti-G value of STAT5.STAT5 is just regulating Bcl-xL to express by the STAT binding member in Bcl-xL promotor.In vivo, in the marrow of the two deficient mice of STAT5A/B-, Bcl-xL is not had to express.And the erythroblast survival of STAT5-mediation depends on the rise of Bcl-xL.In recent years, proved that the transgenosis process LAN of Bcl-xL in mouse B cell can promote that B cell is survived and non-malignant plasmocyte stove.

The inventive method is particularly suitable for producing the celliferous cell culture of RSV specific antibody comprising and can breed with secretory antibody.In one embodiment, RSV specific memory B-cell is used, to produce in vitro B cell culture.Described memory B cell is preferably people's cell, to produce people's antibody.Described B cell preferably derives from individuality, contacted respiratory syncytial virus before described individuality.In one embodiment, utilize means known in the art by peripheral blood sample and/or tonsilla sample separation RSV specific b cells.Such as, by selecting (magnetic bead sorting) B cell marker CD19 and/or CD22 and (subsequently) to select cell surface IgG and/or CD27 and/or negative selection IgM, IgD and/or IgA, memory B cell is separated.In Germinal center B cell, BCL6 expression level is high, and Blimp-1 expression level is low.Naturally growing of antibody secreting cell comprises Blimp-1 up-regulated.Because Blimp-1 suppresses BCL6 to express, so Blimp-1 rise causes BCL6 to lower under its natural environment.In a preferred embodiment of the present invention, Blimp-1 up-regulated, and BCL6 expresses maintenance at least partly.This causes the RSV specific antibody producing co expression BCL6 and Blimp-1 to produce cell.Described RSV specific antibody produces cell and can breed and secrete anti-RSV antibodies, is therefore applicable in vitro B cell and cultivates.In another preferred embodiment, described antibody produced cell is protected apoptosis not to occur by Bcl-xL.RSV specific antibody generation cell of the present invention provides and keeps stable for a long time and the advantage that end differentiation eventually does not occur.Antibody produced cell of the present invention stablizes at least one week, preferably at least one moon, more preferably at least three months, most preferably at least six months.Preferably under CD40L exists, cultivate B cell of the present invention, because CD40L is conducive to the propagation of most of B cell.In one embodiment, compared with Germinal center B cell, BCL6 expresses and maintains substantially identical or higher level, because significant BCL6 expression and Blimp-1 express the antibody produced cell producing and have preferred propagation and antibody generation characteristic and/or stability.In a preferred embodiment, described BCL6 expresses and/or Blimp-1 expresses and expresses along with Bcl-xL, produces preferred propagation and antibody produces characteristic and/or stability.

Therefore, an embodiment provides a kind of and produces the celliferous method of RSV specific antibody, and described cytotostatic at least one week, preferably at least one moon, more preferably at least three months, more preferably at least six months, described method comprised:

-RSV specific memory B-cell is provided;

-improve the expression level of Blimp-1 in described cell; With

-improve and/or maintain the BCL6 expression level in described cell.

Also provide a kind of and produce the celliferous ex vivo approach of RSV specific antibody, described method comprises the expression level improving Blimp-1 in RSV specific memory B-cell, and the BCL6 expression level improving and/or maintain in described cell.Preferably, described BCL6 with Blimp-1 expression level is adjusted to and/or maintains level substantially identical or higher compared with plasmablast.In a preferred embodiment, to transduce described B cell with BCL6 and Bcl-xL.Therefore, also provide a kind of and produce the stable celliferous method of at least trimestral RSV specific antibody, described method comprises:

-provide BCL6 or its Functional portions, derivative and/or analogue to the B cell that can produce RSV specific antibody.

-provide Bcl-xL or its Functional portions, derivative and/or analogue to described B cell; With

-cultivate described B cell.

The nucleotide sequence of coding BCL6 or its Functional portions, derivative and/or analogue is preferably provided to described B cell, and the nucleotide sequence of coding Bcl-xL or its Functional portions, derivative and/or analogue.

Preferably at the compound that can increase Blimp-1 expression, there is the described B cell of lower cultivation in the Zhan Nasi kinases of such as IL-21, IL-2, IL-6, Il-7, IL-10, IL-15, IL-27 or sudden change.Preferred use IL-21, expresses and stabilization of antibodies generation cell because this cytokine is particularly suitable for strengthening Blimp-1 by the inventive method.And in order to improve transduction efficiency, preferably with coding BCL6 and/or Bcl-xL, or the nucleotide sequence of its Functional portions, derivative and/or analogue is transduceed before described B cell, cultivates described B cell under IL-21 exists.

In one embodiment, SOCS albumen or its Functional portions, derivative and/or analogue is provided to described B cell, or their nucleic acid of encoding, expresses because SOCS albumen or its Functional portions, derivative and/or analogue can strengthen Blimp-1 indirectly.In the embodiment that another is optional or extra, provide E47 or its Functional portions, derivative and/or analogue to described B cell, or their nucleic acid of encoding.As previously mentioned, as the result that E47 or its Functional portions, derivative and/or analogue level improve, Socs protein function strengthens, and indirectly improves Blimp-1 expression.

Describe particularly preferred embodiment in an embodiment.According to a particularly preferred embodiment, first under IL-21 exists, cultivate RSV specific b cells.Then, with the nucleic acid of coding BCL6 and the nucleic acid of coding Bcl-xL, transduction reaction is carried out to described B cell.The centrifugal transduction of preferred use (spin transduction).Most preferably, mixing comprises B cell and the virus of at least one nucleic acid interested, and then this mixture centrifugal, to realize high transduction efficiency.After transduction, there is not IL-21 and cultivating B cell 3-5 days, to express BCL6 under there is the condition of IL-4 and L-cell.Then, according to this preferred implementation, again with the nucleic acid of coding BCL6 and the nucleic acid of coding Bcl-xL, transduction reaction is carried out to described B cell.Then, again there is not IL-21 and cultivating B cell 3-5 days, to express BCL6 under there is the condition of IL-4 and L-cell.Then, be separated the cell of expressing BCL6 and Bcl-xL, again give this culture by IL-21, to strengthen propagation and antibody generation.In preferred screening and culturing thing supernatant liquor, the antibody of Bcl-6, Blimp1 and Bcl-XL express cell secretion is in vitro to the neutralising capacity/activity/reactivity of RSV.Preferably by, such as limiting dilution cultivates the antibody produced cell selecting to produce these antibody further.Obtain thus stable while express the RSV specific b cells of BCL6 and Blimp-1.Under the condition of cultivating in vitro, described B cell can be bred and produce antibody during at least six months.

An embodiment provides a kind of the inventive method, and the method also comprises to be selected and/or is separated RSV-specific antibody or its function equivalent.In one embodiment, select and be separated IgM generation cell and IgG generation cell.Preferably, select and/or be separated IgG generation cell.

The RSV-specific antibody produced by the inventive method produces cell and is applicable to the antibody producing anti-RSV.But, in a preferred embodiment, by described cell be separated coding Ig heavy chain and/or light chain gene and at the second cell, such as Chinese Hamster Ovary (CHO) clone or 293 (T) cells.Described the second cell, in this article also referred to as producer's cell, is preferably applicable to commercial antibodies and produces.The propagation of described producer's cell obtains producer's clone that can produce RSV-specific antibody.Preferably, described producer's clone is applicable to producing the compound for people.Therefore, described producer's clone is preferably not such as, containing pathogenic agent, pathogenic microbes.

The inventive method is preferred for producing the antibody produced cell stablizing at least one week, preferably at least one month, more preferably at least three months, more preferably at least six months, to carry out commercial antibodies production.Most preferably, the stable cell lines that can produce monoclonal antibody is created.Preferred utilization by the memory B cell of (such as) sample separation, by select CD19 and/or CD22 (B cell marker) and cell surface IgG and/or CD27 (to mark memory cell) and/or by IgM, IgD and/or IgA are carried out negative selection carry out this kind operate.And, utilize RSV or be derived from the component of RSV, such as RSVF albumen, G-protein and/or SH albumen, in binding tests, select RSV specific antibody to produce cell.Subsequently, according to this kind preferred embodiment, produce co expression Blimp-1 and BCL6 in cell at described RSV specific antibody, generation can the culture of cell of specific binding RSV (component).In another preferred embodiment, also Bcl-xL or its Functional portions, derivative and/or analogue is provided to described B cell.

If only use a kind of memory B cell, then obtain the clone of the present invention producing monoclonal antibody.Also may obtain from the B cell that can produce anti-rsv antibodies can the clone of manufacture order clonal antibody.After producing stable B cell culture by the inventive method, separation can produce the B cell of the antibody of anti-RSV specific antigens, in the second clone, preferably express at least Functional portions from the coding Ig heavy chain of described B cell and/or the gene of light chain.Preferably, in the second clone, express at least Functional portions of at least Functional portions from the Ig heavy chain encoding gene of described B cell and Ig light chain encoding gene.

In one embodiment, the antibody produced cell obtained by the individuality of once contacted RSV, preferably but not necessarily memory B cell can be used for the inventive method.In this way, interested people's antibody may be produced in vitro.

Therefore, also provide a kind of generation can specific binding and/or in and the method for antibody of respiratory syncytial virus, described method comprises:

-utilize the inventive method to produce the antibody produced cell that can produce RSV specific antibody; With

-obtain described antibody produced cell produce antibody.

The antibody of the separation or reorganization obtained by the inventive method is also provided, and the antibody produced cell of separation or reorganization, or its function equivalent.Described antibody preferably includes antibody D25, AM14, AM16 and/or AM23, or its Functional portions, derivative or analogue.

Once after obtaining RSV specific antibody of the present invention generation cell, be preferably manually separated and/or produce at least Functional portions of encode the Ig light chain of described cell and/or the gene of heavy chain.In one embodiment, the nucleotide sequence of the Functional portions at least comprising nucleotide sequence shown in Figure 11, Figure 12, Figure 14 A, Figure 14 B and/or Figure 14 C is provided.Described Functional portions preferably comprises at least one nucleotide sequence shown in Figure 11 D, Figure 12, Figure 14 A, Figure 14 B and/or Figure 14 C.At least one CDR shown in described Functional portions optimized encoding Figure 11 D, Figure 12, Figure 14 A, Figure 14 B and/or Figure 14 C.

Also provide a kind of to be separated, the nucleotide sequence of synthesis or restructuring, described nucleotide sequence comprises and sequence C AGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGATG GTCTCCTGCCAGGCCTCTGGAGGCCCCCTCAGAA, ACTATATTATCAAC, TGGCTACGACAGGCCCCTGGACAAGGCCCTGAGTGGATGGGA, GGGATCATTCCTGTCTTGGGTACAGTACACTACGCACCGAAGTTCCAGGGC, AGAGTCACGATTACCGCGGACGAATCCACAGACACAGCCTACATCCATCTGATCAG CCTGAGATCTGAGGACACGGCCATGTATTACTGTGCGAC G, GAAACAGCTCTGGTTGTATCTACTACCTACCTACCACACTACTTTGACAAC, at least partially at least 70% of TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG and/or CAGGTGCAGCTGGTACAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGAT GGTCTCCTGCCAGGCCTCTGGAGGCCCCCTCAGAAACTATATTATCAACTGGCTAC GACAGGCCCCTGGACAAGGCCCTGAGTGGATGGGAGGGATCATTCCTGTCTTGGGT ACAGTACACTACGCACCGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATC CACAGACACAGCCTACATCCATCTGATCAGCCTGAGATCTGAGGACACGGCCATGT ATTACTGTGCGACGGAAACAGCTCTGGTTGTATCTACTACCTACCTACCACACTAC TTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG, preferred at least 80%, more preferably the sequence of heavy chain of at least 90% homology, described part has at least 15 Nucleotide.Described sequence of heavy chain preferred source is from antibody D25.Described sequence of heavy chain preferably comprises the sequence with sequence at least 70% shown in Figure 11 D, preferably at least 80%, more preferably at least 90% homology.Herein also providing package containing the nucleotide sequence of the separation of the sequence of heavy chain that is made up of any above-mentioned sequence of heavy chain, synthesis or restructuring.

Also provide a kind of to be separated, the nucleotide sequence of synthesis or restructuring, described nucleotide sequence comprises and sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGACAGAGT CACCATCACTTGC, CAGGCGAGTCAGGACATTGTCAACTATTTAAAT, TGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAC, GTTGCATCCAATTTGGAGACA, GGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTAGTCTCACCAT CAGCAGCCTGCAGCCTGAAGATGTTGCAACATATTATTGT, CAACAATATGATAATCTCCCA, at least partially at least 70% of CTCACATTCGGCGGAGGGACCAAGGTTGAGATCAAAAGA and/or GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCAGCTGTAGGAGACAGAGT CACCATCACTTGCCAGGCGAGTCAGGACATTGTCAACTATTTAAATTGGTATCAAC AGAAACCAGGGAAAGCCCCTAAGCTCCTGA TCTACGTTGCATCCAATTTGGAGACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGA TCTGGGACAGATTTTAGTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAAC ATATTATTGTCAACAATATGATAATCTCCCACTCACATTCGGCGGAGGGACCAAGG TTGAGATCAAAAGA, preferred at least 80%, more preferably the sequence of light chain of at least 90% homology, described part has at least 15 Nucleotide.Described sequence of light chain preferred source is from antibody D25.

Described sequence of light chain preferably comprises the sequence with sequence at least 70% shown in Figure 11 D, preferably at least 80%, more preferably at least 90% homology.Herein also providing package containing the nucleotide sequence of the separation of the sequence of heavy chain that is made up of any above-mentioned sequence of light chain, synthesis or restructuring.

Also provide a kind of to be separated, the nucleotide sequence of synthesis or restructuring, described nucleotide sequence comprises and sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG ACTCTCCTGTGCGGCCTCT, GGATTCAGCTTCAGTCACTATGCC, ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTT, ATATCTTATGATGGAGAAAATACA, TATTACGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTCCAAGAA CACAGTGTCTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTCTATATTACT GT, GCGAGAGACCGCATAGTGGACGACTACTACTACTACGGTATGGACGTC, at least partially at least 70% of TGGGGCCAAGGGGCCACGGTCACCGTCTCCTCAG and/or GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG ACTCTCCTGTGCGGCCTCTGGATTCAGCTTCAGTCACTATGCCATGCACTGGGTCC GCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTTATATCTTATGATGGAGAA AATACATATTACGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATTC CAAGAACACAGTGTCTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTCTAT ATTACTGTGCGAGAGACCGCATAGTGGACGACTACTACTACTACGGTATGGACGTC TGGGGCCAAGGGGCCACGGTCACCGTCTCCTCA, preferred at least 80%, more preferably the sequence of heavy chain of at least 90% homology, described part has at least 15 Nucleotide.Described sequence of heavy chain preferred source is from antibody A M14.Herein also providing package containing the nucleotide sequence of the separation of the sequence of heavy chain that is made up of any above-mentioned sequence of heavy chain, synthesis or restructuring.

Also provide a kind of to be separated, the nucleotide sequence of synthesis or restructuring, described nucleotide sequence comprises and sequence GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGT CACCATCACTTGCCAGGCGAGT, CAGGACATTAAGAAGTAT, TTAAATTGGTATCATCAGAAACCAGGGAAAGTCCCTGAGCTCCTGATGCAC, GATGCATCC, AATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGCAGGGGATCTGGGACAGATTT TACTCTCACCATTAGCAGCCTGCAGCCTGAAGATATTGGAACATATTACTGT, CAACAGTATGATAATCTGCCTCCGCTCACT, at least partially at least 70% of TTCGGCGGAGGGACCAAGGTGGAGATCAAAC and/or GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGT CACCATCACTTGCCAGGCGAGTCAGGACATTAAGAAGTATTTAAATTGGTATCATC AGAAACCAGGGAAAGTCCCTGAGCTCCTGATGCACGATGCATCCAATTTGGAAACA GGGGTCCCATCAAGGTTCAGTGGCAGGGGATCTGGGACAGATTTTACTCTCACCAT TAGCAGCCTGCAGCCTGAAGATATTGGAACATATTACTGTCAACAGTATGATAATC TGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTG, preferred at least 80%, more preferably the sequence of light chain of at least 90% homology, described part has at least 15 Nucleotide.Described sequence of light chain preferred source is from antibody A M14.Herein also providing package containing the nucleotide sequence of the separation of the sequence of heavy chain that is made up of any above-mentioned sequence of light chain, synthesis or restructuring.

Also provide a kind of to be separated, the nucleotide sequence of synthesis or restructuring, described nucleotide sequence comprises and sequence GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCT, GGATTCACATTCAGTAGTTATAAC, ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACAC, ATTAGTGCGGGTAGTAGTTACATA, TACTACTCAGACTCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGTCAGGAA CTCAGTATATCTGCAAATGAACAGCCTGAGAGCCGCTGACACGGCTGTGTATTACT GT, GCGAGAGAGGATTATGGTCCGGGAAATTATTATAGTCCTAACTGGTTCGACCCC, at least partially at least 70% of TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG and/or GAGGTGCAGCTGGTGGAGACCGGGGGAGGCCTGGCCCAGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACATTCAGTAGTTATAACATGAACTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACACATTAGTGCGGGTAGTAGT TACATATACTACTCAGACTCAGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGT CAGGAACTCAGTATATCTGCAAATGAACAGCCTGAGAGCCGCTGACACGGCTGTGT ATTACTGTGCGAGAGAGGATTATGGTCCGGGAAATTATTATAGTCCTAACTGGTTC GACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA, preferred at least 80%, more preferably the sequence of heavy chain of at least 90% homology, described part has at least 15 Nucleotide.Described sequence of heavy chain preferred source is from antibody A M16.Herein also providing package containing the nucleotide sequence of the separation of the sequence of heavy chain that is made up of any above-mentioned sequence of heavy chain, synthesis or restructuring.

Also provide a kind of to be separated, the nucleotide sequence of synthesis or restructuring, described nucleotide sequence comprises and sequence C AGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGAGTCACC ATCTCCTGCACTGGGAGC, AGCTCCAACATCGGGGCAGGTTATGAT, GTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTAT, GGCAACACT, AATCGGCCCTCAGGGGTCTCCGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGC CTCCCTGGCCATCACTGGACTCCAGGCTGAGGATGAGGCTGATTATTACTGC, CACTCCTATGACAGAAGCCTGAGTGGT, at least partially at least 70% of TCAGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAG and/or CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGAGTCAC CATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAG GTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATC TATGGCAACACTAATCGGCCCTCAGGGGTCTCCGACCGATTCTCTGGCTCCAAGTC TGGCACCTCAGCCTCCCTGGCCATCACTGGACTCCAGGCTGAGGATGAGGCTGATT ATTACTGCCACTCCTATGACAGAAGCCTGAGTGGTTCAGTATTCGGCGGAGGGACC AAGCTGACCGTC, preferred at least 80%, more preferably the sequence of light chain of at least 90% homology, described part has at least 15 Nucleotide.Described sequence of light chain preferred source is from antibody A M16.Herein also providing package containing the nucleotide sequence of the separation of the sequence of heavy chain that is made up of any above-mentioned sequence of light chain, synthesis or restructuring.

Also provide a kind of to be separated, the nucleotide sequence of synthesis or restructuring, described nucleotide sequence comprises and sequence C AGGTGCAACTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTGAGA CTGTCCTGTGCAGCGACT, GGATTCAACTTCCATAACTACGGC, ATGAACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGGTT, GTTTGGTATGATGGAAGTAAGAAA, TACTATGCAGACTCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGAA CACTCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTTTATTATT GT, GTGAGAGATAAAGTGGGACCGACTCCCTACTTTGACTCC, at least partially at least 70% of TGGGGCCAGGGAACCCTGGTCACCGTATCCTCAG and/or GAGGTGCAGCTGGTGGAGTCTGGGGGAAATGTGGTCAAGCCTGGGACGTCCCTGAG ACTGTCCTGTGCAGCGACTGGATTCAACTTCCATAACTACGGCATGAACTGGGTCC GCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGGTTGTTTGGTATGATGGAAGT AAGAAATACTATGCAGACTCCGTGACGGGCCGATTCGCCATCTCCAGAGACAATTC CAAGAACACTCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTTT ATTATTGTGTGAGAGATAAAGTGGGACCGACTCCCTACTTTGACTCCTGGGGCCAG GGAACCCTGGTCACCGTCTCGAGT, preferred at least 80%, more preferably the sequence of heavy chain of at least 90% homology, described part has at least 15 Nucleotide.Described sequence of heavy chain preferred source is from antibody A M23.Herein also providing package containing the nucleotide sequence of the separation of the sequence of heavy chain that is made up of any above-mentioned sequence of heavy chain, synthesis or restructuring.

Also provide a kind of to be separated, the nucleotide sequence of synthesis or restructuring, described nucleotide sequence comprises and sequence TCCTATGTGCTGACTCAGCCACCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCGC GATCACCTGTGGAAGAAAC, AACATTGGAAGTGAAACT, GTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTAT, GATGATGAC, GACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGC CACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTACTGT, CAGGTGTGGGATAGGAGTAATTATCATCAGGTA, at least partially at least 70% of TTCGGCGGAGGGACCAAGTTGACCGTCCTAG and/or TCCTATGTGCTGACTCAGCCCCCCTCGGTGTCACTGGCCCCAGGAGGGACGGCCGC GATCACCTGTGGAAGAAACAACATTGGAAGTGAAACTGTGCACTGGTACCAGCAGA AGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATGACGACCGGCCCTCAGGG ATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAG CAGGGTCGAGGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGGAGTA ATTATCATCAGGTATTCGGCGGAGGGACCAAGCTGACCGTC, preferred at least 80%, more preferably the sequence of light chain of at least 90% homology, described part has at least 15 Nucleotide.Described sequence of light chain preferred source is from antibody A M23.Herein also providing package containing the nucleotide sequence of the separation of the sequence of heavy chain that is made up of any above-mentioned sequence of heavy chain, synthesis or restructuring.

Also provide the nucleotide sequence of the Functional portions at least 70% of coding and at least aminoacid sequence shown in Figure 11, Figure 14 A, Figure 14 B and/or Figure 14 C, preferred at least 80%, more preferably at least 90% identical aminoacid sequence, described part has at least 5 amino-acid residues.The aminoacid sequence that described nucleotide sequence optimized encoding is identical with the heavy CDR sequences 1,2 and/or 3 shown in Figure 11 D and/or CDR sequence 1 or 2 at least 80%.In another preferred embodiment, the aminoacid sequence that at least one CDR sequence at least 80% shown in described nucleic acid sequence encoding with Figure 14 A, Figure 14 B and/or Figure 14 C is identical.In a preferred embodiment, sequence of heavy chain shown in described nucleic acid sequence encoding with Figure 11 A, with Figure 14 A shown in sequence of heavy chain, with Figure 11 B shown in sequence of heavy chain, with Figure 14 C shown in sequence of heavy chain, with Figure 11 A shown in sequence of light chain, with Figure 14 A shown in sequence of light chain, with Figure 14 B shown in sequence of light chain and/or the aminoacid sequence identical with sequence of light chain at least 70% shown in Figure 14 C.

Therefore, also provide the nucleotide sequence of a kind of separation, synthesis or restructuring, described nucleotide sequence comprises the sequence of coding and aminoacid sequence at least 70% shown in Figure 11 A-D, preferred at least 80%, more preferably at least 85% identical aminoacid sequence.The aminoacid sequence that described nucleotide sequence optimized encoding is identical with the heavy CDR sequences 1,2 and/or 3 shown in Figure 11 A-D and/or CDR sequence 1 or 2 at least 80%.An embodiment provides a kind of and is separated, the nucleotide sequence of synthesis or restructuring, it is identical with aminoacid sequence NYIIN at least 70% that described nucleotide sequence comprises coding, and/or it is identical with sequence GIIPVLGTVHYAPKFQG at least 75%, and/or it is identical with sequence ETALVVSTTYLPHYFDN at least 70%, and/or it is identical with sequence QASQDIVNYLN at least 85%, and/or it is identical with sequence VASNLET at least 70%, and/or it is identical with sequence QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLG TVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHY FDN WGQGTLVTVSS at least 70%, and/or the sequence of the aminoacid sequence identical with sequence D IQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETG VPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTV at least 70%.

Nucleotide sequence of the present invention preferably with any above-mentioned sequence at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% homology.

Also provide the nucleotide sequence of a kind of separation, synthesis or restructuring, described nucleotide sequence comprises the sequence of coding and aminoacid sequence at least 70% shown in Figure 14 A-C, preferred at least 80%, more preferably at least 85% identical aminoacid sequence.The aminoacid sequence that described nucleotide sequence optimized encoding is identical with CDR sequence at least 70% shown in Figure 14 A, 14B and/or 14C.An embodiment provides a kind of and is separated, the nucleotide sequence of synthesis or restructuring, described nucleotide sequence comprises the sequence of following aminoacid sequence of encoding, and described aminoacid sequence is identical with the aminoacid sequence at least 70% being selected from lower group: GFSFSHYA, ISYDGENT, ARDRIVDDYYYYGMDV, QDIKKY, DAS, QQYDNLPPLT, EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGE NTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDV WGQGATVTVSS, DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLET GVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGGTKVEIKRTV, GFTFSSYN, ISAGSSYI, AREDYGPGNYYSPNWFDP, SSNIGAGYD, GNT, HSYDRSLSG, EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHISAGSS YIYYSDSVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNWF DPWGQGTLVTVSS, QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNR PSGVSDRFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGSVFGGGTKLTV, GFNFHNYG, VWYDGSKK, VRDKVGPTPYFDS, NIGSET, DDD, QVWDRSNYHQV, EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWVRQAPGKGLEWVAVVWYDGS KKYYADSVTGRFAISRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVGPTPYFDSWGQ GTLVTVSS and SYVLTQPPSVSLAPGGTAAITCGRNNIGSETVHWYQQKPGQAPVLVVYDDDDRPSG IPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSNYHQVFGGGTKLTV.

As described above, nucleotide sequence of the present invention is particularly suitable in nucleic acid expression systems, express antibody of the present invention or its Functional portions, derivative or analogue, preferred D25, AM14, AM16, AM23 or its Functional portions, derivative and/or analogue.Nucleotide sequence of the present invention preferably at cells, more preferably at producer's cells of applicable antibody producing.

Explain the present invention further in the examples below.These embodiments do not limit the scope of the invention, only for setting forth the present invention.

Embodiment

Materials and methods

Maintain and be separated human B cell

Use standard method, from the dark yellow layer separation of C D19 positive human B cell (other sources can be the fresh bloods containing anticoagulant factor, or lymphoid organ amygdala or spleen) being derived from blood storehouse.Briefly, phenanthrene is utilized (ficoll) density fractionation (the An Ma West Asia company (Amersham, Buckinghamshire, UK) of Britain's Buckinghamshire) total peripheral blood lymphocytes (PBMC) can be separated.The MACS cell sorting techniques (the close special company (Miltenyi, Utrecht, Netherlands) of Utrecht, Netherlands) described by manufacturer, the pearl utilizing CD22 to mark just selects B cell.Utilize monoclonal antibody (mAb) (BD company (the Becton Dickinson in lake, New Jersey Franklin of CD19, CD27, IgD, IgM and IgA subsequently, BD, Franklin Lakes, NJ, USA)) appropriately combined to cell dyeing.Then, positive with FACSAria (BD) sorting CD19 and CD27 and the memory B cell (Fig. 1) of IgM, IgA and IgD feminine gender.Except memory B cell, other B cell subgroups can be separated, such as initiating cell, follicular cells, memory cell, antibody produced cell, center parent cell, centrocyte, germinal center cells, plasmablast, plasmocyte, marginarium cell, perisinusoidal cell or B cell of dividing a word with a hyphen at the end of a line (in these subgroups, many subgroups only measured in mouse) with suitable marker.

Cell cultures

The cell of washing sorting, and be covered with the L cell (5x10 of the expression CD40L irradiated through 80 Grays ⁴individual cells/ml; By French Xian Lin Schering-Plough (Schering Plough France, DardillyFrance) doctor J.Banchereau provides) 24 orifice plates on, use perfect medium (the D minimum essential medium of the Yi Kefu improvement containing 8% foetal calf serum (FCS) and penicillin/streptomycin (Iscove ' s Modified D Minimal Essential Medium)) cultivation (1.5-2x 10 ⁵individual cells/ml).Except as otherwise noted, these L cells of expressing CD40L are always by the culture medium culturing comprising 8%FCS.In order to the B cell for the preparation of retroviral transduction, at mouse IL-21 (50ng/ml, R & D company (the R & D of Minn. Mi get Lan Minneapolis, Minneapolis, MN, USA)) there are lower culturing cell 36 hours.After transduction, preferably culturing cell under IL-21 exists, but cell has response to IL-4, IL-15 and IL-10 (not getting rid of other cytokines).Such as, the B cell amplification of IL-4 induction, lower than IL-21, may need lower cell fission level in some experiments.

The generation of retroviral construct thing and recombinant retrovirus

Recorded the constitutive activity mutant of STAT5a and b in the past.Encode the DNA of these mutant and wild-type STAT5b available from T.Kitamura (the IMSUT company (IMSUT, Tokyo, Japan) of Tokyo).In the aging rescue screening of mouse fibroblast cell, Bcl-6 is accredited as the inhibitor of antiproliferative p19ARF-p53 signal transduction.Bcl-XL is accredited as anti-apoptosis factor, and it is provided by doctor Korsmeyer (the HH Institute for Medical Research (Howard Hughes Medical Institute, Boston, US) of boston, u.s.a) friendship.(Heemskerk etc., 1997 in LZRS-joint-IRES-GFP (or IRES-YFP or the IRES-NGFR) carrier recorded before these DNA are connected to; Heemskerk etc., 1999).Also IRES-YFP (yellow fluorescence protein) or IRES-NGFR (trk C) can be used to substitute IRES-GFP (green fluorescent protein) marker.NGFR is the signal transduction offunction mutations body of NGFR, is provided by doctor's C.Bonini friendship.The monoclonal antibody (the Crow Ma Pu company (Chromaprobe, Mountain View, CA, US) of California, USA Mountain View or close special company (Miltenyi)) of NGFR is used to observe the cell of expression of NGF R.

In order to produce recombinant retrovirus, use Fugene-6 (Roche Diagnistics dutch company (the Roche Diagnostics Netherlands of Dutch A Er mil, Almere, Netherlands)), according to manufacturer's scheme, retroviral plasmid is transfected in complementary virus-free amphophilic producer clone Phoenix-A, this clone is derivative (Kinsella and Nolan of human embryonic kidney cell line 293, 1996) (by the doctor G.Nolan (Dr.G.Nolan of the Stanford University of Palo Alto, CA, Stanford University, Palo Alto, CA) friendship present).Two days later, start to select the cell of transfection by adding 2 μ g/ml tetracyclines (BD of Palo Alto, CA clones Tyke Laboratories, Inc (Becton Dickinson Clontech Laboratories)).10-14 days after transfection, by 6x 10 ⁶cell is inoculated into (BD discovery laboratory equipment company (the Becton Dickinson Discovery Labware of Massachusetts Bedford in the petri diss of a 10cm, Bedford, MA)), the perfect medium wherein containing 10ml not purine-containing mycin.Second day, change fresh culture, the 3rd day, results Retroviral supernatant, centrifugal and frozen in-70 DEG C with acellular sample aliquot form.This method provides a kind of reproducible quick, extensive and high-titer retrovirus production method, and its output is more than 3x 10 ⁶individual infectious viral particle/milliliter.

Retroviral transduction

As recorded (Heemskerk etc., 1997 in the past; Heemskerk etc., 1999) carry out recombinant human fibronectin polypeptide fragment CH-296 transduction method (RetroNectin ^tM; The Taka in the large Tianjin of Japan draws company (Takara, Otsu, Japan)).Spend the night by 0.3ml 30 μ g/ml recombinant human fibronectin polypeptide fragment CH-296 room temperature treatment 2 hours or 4 DEG C of process, to wrap by 24 orifice plates of the non-tissue culture treated (Ke Sita company (Costar of Dutch Badhoevedorp, Badhoevedorp, Netherlands)).When using the lung tissue culture plate of different size, use reagent in proportion.Take out CH-296 solution, together with 2% human serum albumin (HSA) then at room temperature prepared with phosphate-buffered saline (PBS), cultivate 30 minutes, then wash once with PBS.By the 5x10 prepared for retroviral transduction ⁵individual B cell is inoculated into not containing in the 0.25ml RPMI of FCS and L-cell, and the Retroviral supernatant melted with 0.25ml mixes.For the two transduction of Bcl-6Bcl-XL, mix 125 μ l Bcl-6-IRES-NGFR (or IRES-YFP) (Shvarts A. etc., Genes Dev., 2002) and 125 μ l Bcl-XL-IRES-GFP (HH Institute for Medical Research (the Howard Hughes Medical Institute of boston, u.s.a children's hospital, Childrens Hospital, Boston, USA) S.Korsmeyer provide), and to add in cell.Then, this culture with 1800rpm, 25 DEG C centrifugal 60 minutes, 37 DEG C cultivate 6 hours.Next, take out 0.25ml supernatant liquor, add the Retroviral supernatant that 0.25ml is fresh.This culture again with 1800rpm, 25 DEG C centrifugal 60 minutes, 37 DEG C of cultivations are spent the night.Morning, cell is transferred in 24 hole tissue culturing plates (Ke Sita company (Costar)), there is people IL-4 (50ng/ml) or mouse IL-21 (50ng/ml, R & D company (the R & D of Minn. Minneapolis, Minneapolis, MN, USA)) normal condition under cultivate 3-5 days.By signal transduction offunction mutations body (the Δ NGFR of the trk C of brachymemma, by Milan, ITA Sheng Lafuer hospital (St.Raphael Hospital, Milan, Italy) C.Bonini provide) antibody staining or GFP and/or YFP (being total to) express determine transduction efficiency.Select to carry out further experiment containing genetically modified cell interested.

Flow cytometry

By FITC, PE, PERCP, PE-Cy5, the antibody of people's molecule I gD, IgG, CD3, CD19, CD20, CD27, CD38, CD40, CD45, CD56, CD70, CD80, CD86, HLA-DR (BD) that APC or APC-Cy7 directly marks, and IgM, κ light chain directly marked with PE (DAKO), lambda light chain, CD138 are used for flow cytometry.Staining cell is analyzed, by FlowJo computer software (TS company (Tree Star, Inc.)) processing FACS data with LSRII (BD).

Proliferation experiment

Original and memory B cell is separated by the fresh PBMC on FACSAria:

Origi-nal B-cell: CD19-Pe-Cy7pos, CD27-APC neg, IgD-PE pos

Memory B cell: CD19-Pe-Cy7pos, CD27-APC pos, IgD-PE neg, IgA-FITC neg

Use PBS washed cell, be resuspended in 0.5ml not containing the RPMI (37 DEG C) of FCS.The IMDM containing 2 μMs of Fluoresceincarboxylic acid succinimido esters (CFSE) of equivalent is added in cell mixture, cultivates 7 minutes for 37 DEG C.With ice-cold FCS washed cell, to stop the mark of cell.Cell is resuspended in 500 μ l IMDM-8%FCS, and cultivates together with L cell when presence or absence IL-21.Unlabeled cells is with comparing.

After 36 hours (just before transduction), analyze the CFSE content of a part of cell.With all the other cells of the centrifugal transduction of Bcl-6-IRES-NGFR, cultivate 3 days, analyze its CFSE content with LSRII.With FlowJo software (TS company) analytical data.

Antigen-specific human B cell is separated with the unicellular sorting of high speed

Except the memory B cell partition method of above-mentioned from MBC (i.e. 100 cell/culture hole), also the antigen of using fluorescence mark is cultivated people's memory B cell and carries out sorting according to antigen recognition.Example is the B cell (being provided by the A.Radbruch (A.Radbruch, Berlin, Germany) of Berlin, Germany) (Fig. 4) of the Toxoid,tetanus that separation and combination phycoerythrin (PE) marks.With 1 cells/well culturing cell, and check that TT combines.But, the antigen that any other marks can be used.

The B-cell receptor (BCR) that the long-term cultivation measuring Bcl-6 and Bcl-XL transducer cell causes is expressed and is changed

In known B cell of breaking up between incubation period in vitro, the film expression of BCR disappears, in the B cell that EBV transforms, also observe this phenomenon.Therefore, for Bcl-6 and Bcl-XL transduction and IL-21 exist under cultivate B cell carry out GFP, NGFR, CD19, κ and/or λ or IgG dyeing, or with mark Toxoid,tetanus dye.In order to prove the availability that BCR expresses, we with FACSAria (BD) with 1 cells/well (96 orifice plate) sorting TT-PE (Radbruch) in conjunction with cell, be then inoculated in the substratum containing L cell and IL-21.After three weeks, detect with FACS Canto (BD) Toxoid,tetanus growing clone and combine.So far, harvested cell and in 96 orifice plates with GFP, NGFR, CD19 and TT-PE dyeing.

The two positive B-cells system of Bcl-6 and Bcl-XL of exploitation secretory antibody

Foundation can produce monoclonal antibody and be the two positive B cell system of 100%Bcl-6 and Bcl-XL.First, this purpose is realized with IL-21 proliferative induction and differentiation.Meanwhile, with these cells of Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFP retroviral transduction.IL-4 maintains these cells 3-4 days.Subsequently, this transgenosis of cell expressing of wherein one or both retroviral transduction is used, thus expression of NGF R or GFP albumen.LSRII (BD) can be utilized to observe the expression of NGFR and/or GFP.If necessary, can transducer cell again, to obtain expression two kinds of genetically modified cells of greater amt.No matter whether carry out second time transduction, FACS Aria (BD) sorting is all used to express two kinds of genetically modified cells, cultivate with 96 orifice plates under the condition that there is IL-21 and 2500-5000L cells/well, cell density scope is 10-500 cells/well.By the 5th day, these short run cultures (MBC) secreted considerable antibody in culture supernatants, and these antibody can be used for screening object subsequently.Screening can based on the techniques available of antigen interested, such as ELISA/EIA/RIA, western blot or direct function experiment, as in and cytokine enclosed experiment.After the MBC of Selection and screening identification antigen interested (being TT and RSV in our experiment), with the density of 0.5 –, 1 cells/well subcloned cells in 96 orifice plates that there is IL-21.Subclone carries out 2-3 week usually, is undertaken by limiting dilution (LD) culture or flow cytometry (FACSAria) unicellular sorting.

RSV A-2 virus stocks and HEp2 clone

Mass propgation RSV A-2 virus (props up WKZ (WKZ by Utrecht, Utrecht) G.van Bleek friendship provides) and HEp2 clone (clinical labororatory (the Clinical Laboratory of Amsterdam AMC, AMC,), and frozen in liquid nitrogen Amsterdam).

In T175Falcon culturing bottle, cultivate HEp2 attached cell system with normal incubation medium, then frozen sample aliquot.

In order to obtain the RSV mother liquor of more efficient valency, inoculation HEp2 cell is also cultivated and is reached 50-60% and be paved with.45 ' is at room temperature utilized to be added on HEp2 cell by original RSV mother liquor (1/20 dilution, cumulative volume 5ml).Adding 15ml fresh culture, cell being put into 37 DEG C, 5%CO when opening bottle cap ₂, o/n.Morning, carefully removes culture supernatants, adds the substratum that 15ml contains 1%FCS.Screw bottle cap, cell is put into 37 DEG C, 5%CO ₂in hatch 24-36 hour.When the synplasm of RSV induction is high-visible, most of synplasm has still kept, and collects substratum, and filter (0.22 μm), at room temperature 1450rpm is centrifugal, then quick freeze sample being stored in liquid nitrogen.By add new substratum containing 1%FCS immediately and after 4-6 hour freezing acquisition second gleanings.

For the RSV lysate of ELISA

The virus stocks infecting the acquisition of HEp2 cell with RSV A-2 can be used for being separated rsv protein.Carefully wash first hole with PBS, and use tryptic digestion.Wash trypsin Ji Buke company (Gibco) off), with 1% octyl glucoside lysing cell precipitation (the cell precipitation 2ml octyl glucoside process of a T175 culturing bottle).With syringe and syringe needle homogenization suspension (upper and lower 10 times), incubated on ice 1 hour, then dialyse by 2L TBS pH of buffer 7.4, o/n at 4 DEG C.After centrifugal removing cell debris, obtain supernatant liquor.Mensuration protein content is 3.6mg/ml, is used for ELISA with the concentration of 20 μ g/ml (50 μ l).

Measure the TCID of RSV mother liquor ₅₀and PFU

In order to measure TCID50, by 10 ⁴individual HEp2 is inoculated in 96 orifice plates, infects HEp2 by the RSV virus of 2 or 10 step serial dilutions with 4-plo.After 2-3 days, remove culture supernatants, at room temperature use 80% acetone fixed cell 10 '.After removing acetone, dry fixing cellular layer, keeps 4 DEG C or frozen in-20 DEG C.In order to RSV HEp2 cell dyeing, first with 5% milk powder blocking of plates of PBS 0.1% polysorbas20 preparation.Then dull and stereotyped 3 times of washing, then cultivates 3-5 hour at 37 DEG C together with the anti-RSV-HRP of polyclonal goat (1:500, the biological design corporation (Biodesign, Saco, ME, US) of maine state of u.s.a Mermithidae), thoroughly washs.Next, at room temperature this hole 30 ' is hatched with AEC substrate.The stove point infected dyes redness, counts by opticmicroscope visual inspection.TCID determined by use standard Excel software ₅₀.

In order to determine plaque forming unit (PFU) numerical value of virus, in 24 orifice plates, by 1x10 ⁵/ mlHEp2 cell and the nutrient solution 10 times of serial dilutions (10 used containing 1%FCS ^-3– 10 ^-7) RSV virus stocks one arise from 37 DEG C and cultivate 45 ' (200 μ l), then use 0.25% extra large spot (seaplaque) agar (Bai Zai enzyme company (Biozyme)) of 0.5ml hand temperature to cover cell and virus.Agarose layer can prevent virus to be disseminated on non-infected cells by substratum.Thus, virus can only infect flanking cell, and these cells are finally killed by virus, in HEp2 cell monolayer, form plaque.With 1% crystal violet solution to the dyeing of fixing cell (96% Yi Chun – 100% Yi Suan – 10% formaldehyde 6:2:1), to observe these plaques.Plaque is counted (at least being counted by two Different Individual), determines PFU numerical value.

The selection of respiratory syncytial virus (RSV) neutralizing antibody

In order to obtain anti-respiratory syncytial virus (RSV) B cell clone, be separated the peripheral blood cells (PBMC) (donor B62 and B63) of two donors from the dark yellow layer being derived from blood storehouse.With FACSAria (BD) sorting CD19 ^posigM ^negigD ^negigA ^negcD27 ^posbefore cell (Fig. 1), with MACS pearl and post (close special company) separation of C D22+ cell.Cell is cultivated together with L cell, except as otherwise noted.Culturing cell 36 hours under IL-21 exists, then only transduces with Bcl-6-IRES-NGFR.12 h before harvest cells, cultivate 3 days under IL-4 exists, and then use MACS pearl (close special company) sorting NGFR express cell and transduce with Bcl-XL-IRES-GFP immediately.Washing not in conjunction with the B cell of MACS pearl, and is transduceed with Bcl-6 and Bcl-XL simultaneously.12 h before harvest cells, collect and cultivate 3 days under IL-4 exists, and then express according to GFP and NGFR on FACSAria and carry out sorting.Washed cell, and under IL-21 exists, cultivate in 96 orifice plates (Ke Sita company (Costar)) with the density of 100 cells/well.

Utilize the HEp2 cell lysate ELISA infecting RSV to screen the RSV of Bcl-6 and Bcl-XL two transduction B cell culture in conjunction with situation, carry out Parallel testing with RSV microneutralization experiment.Briefly, by 10 ⁴individual HEp2 cell is inoculated in flat 96 orifice plates (Ke Sita company) containing perfect medium.Second day, at room temperature replace substratum 1 hour with the mixture of 37 DEG C of preincubates RSV of 30 minutes virus and cell culture supernatant.Cumulative volume is 25 μ l, and RSV end level is 0.1MOI.After 1 hour, with PBS by vial supernatant mixture diluted 9 times, and replace with 100 μ lIMDM/5%FCS.After 2 days, with 80% acetone fixed cell, and dye with Anti-TNF-α-RSV-HRP (biological design corporation (Biodesign)).Use H ₂o ₂and AEC, the cell infecting RSV produces red.The cell using observation by light microscope to infect, if necessary counts.Use the contrast that the anti-RSV of Goat polyclonal (the Abcam company in Cambridge, Massachusetts (Cambridge, MA)) neutralizes as RSV.

The RT-PCR in VH and VL district and clone

With trace quantity reagent kit (the Kai Jie company (Qiagen, Venlo, The Netherlands) of Dutch fragrant network) is from about 5x10 ⁵individual B cell is separated total serum IgE.Reverse transcription 250ng total serum IgE in the system of 1X first chain damping fluid, 500 μMs of dNTP, 250ng random hexamers, 5mM DTT, 40U RNasin (Pu Luomaige company (Promega)) and 200U SuperScript III RT (hero company (Invitrogen)) is contained at 20 μ l.With ultrapure water, cDNA is diluted 10X, contain 20mM Tris-HCL, 50mM KCL, 2.5mM MgCl at 50 μ l ₂, 250 μMs of dNTP, 1U AmpliTaq gold mark archaeal dna polymerase (Applied Biosystems, Inc. (Applied Biosystems Inc.)) and each primer of 25pmol solution in PCR is carried out to 2.5 μ l cDNA.PCR condition is as follows: 96 DEG C are carried out 8 minutes denaturing steps, is then 35 following circulations: 96 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 1 minute; Be finally 72 DEG C and extend 10 minutes.

According to manufacturer's suggested design, electrophoresis PCR primer on sepharose, purifying is also cloned in pCR2.1TA cloning vector.Sequential analysis is carried out with BigDye terminator chemistry (Applied Biosystems, Inc.) and Vector-NTI software (hero company).

In order to get rid of the sudden change that reversed transcriptive enzyme and/or archaeal dna polymerase bring out, carry out some items independently cDNA to transform and PCR reacts, and carry out independent Cloned culturing.With Vector-NTI contig express (Vector-NTI Contig Express) software determination consensus sequence.

In order at 293T cells recombinant protein antibody, produce total length heavy chain and light chain construct with pCDNA3.1 (+) Zeo (hero company).By heavy chain leader and the VH district of pcr amplification clone D25, introduce 5 '-NheI site and 3 '-XhoI site, thus build heavy chain expression vector.By same cDNA amplification IgG1 constant region (CH1-hinge-CH2-CH3), introduce 5 '-XhoI and 3 '-NotI site simultaneously.PCDNA3.1 (+) Zeo being connected into NheI/NotI digestion by 3 obtains total length heavy chain expression vector.With primer by pcr amplification light chain leader, VL district and constant region of light chain, introduce 5 '-NheI and 3 '-NotI site, thus produce full-length light chains expression constructs.Full-length light chains expression vector is obtained in pCDNA3.1 (+) Zeo rear product cloning digested to NheI/NotI.

Carry out sequential analysis, to confirm the exactness of this expression constructs.

With heavy chain and light chain expression vector, instantaneous dual-transfected (Fugene-6 is carried out to 293T cell, Germany Roche Holding Ag (Roche, or lipofectin LTX (Lipofectamine LTX) Germany), hero company), thus obtain recombinant monoclonal antibodies.FACS dyeing (48 hours) is carried out, to show the functional combination of antibody and RSV F-albumen by the culture supernatants of the Hep2 cell infecting RSV.

Oligonucleotide for pcr amplification is:

vH district:

VH1-forward 5 '-AAATCGATACCACCATGGACTGGACCTGGAGG-3'

VH1B-forward 5 '-AAATCGATACCACCATGGACTGGACCTGGAGM-3'

VH2A-forward 5 '-AAATCGATACCACCATGGACACACTTTGCTMCAC-3'

VH2B-forward 5 '-AAATCGATACCACCATGGACATACTTTGTTCCAAC-3'

VH3-forward 5 '-AAATCGATACCACCATGGAGTTTGGGCTGAGC-3'

VH3B-forward 5 '-AAATCGATACCACCATGGARYTKKGRCTBHGC-3'

VH4-forward 5 '-AAATCGATACCACCATGAAACACCTGTGGTTCTT-3'

VH5-forward 5 '-AAATCGATACCACCATGGGGTCAACCGCCATC-3'

VH6-forward 5 '-AAATCGATACCACCATGTCTGTCTCCTTCCTC-3'

C γ-oppositely 5'-GGGTCTAGACAGGCAGCCCAGGGCCGCTGTGC-3'

v κ district:

Vk1-forward 5 '-AAATCGATACCACCATGGACATGAGGGTCCCY-3 '

Vk1B-forward 5 '-AAATCGATACCACCATGGACATGAGRGTCCYY-3 '

Vk2-forward 5 '-AAATCGATACCACCATGAGGCTCCCTGCTCAG-3 '

Vk3-forward 5 '-AAATCGATACCACCATGGAARCCCCAGCGCA-3 '

Vk4-forward 5 '-AAATCGATACCACCATGGTGTTGCAGACCCAG-3 '

The reverse 5 '-GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT-3 ' of Ck-

v λ district:

Vl1aecb 5’-AAATCGATACCACCATGGCCTGGTCCCCTCTCCTCC-3’

Vl1g 5’-AAATCGATACCACCATGGCCGGCTTCCCTCTCCTCC-3’

Vl2/10 5’-AAATCGATACCACCATGGCCTGGGCTCTGCTCCTCC-3’

Vl3jpah 5’-AAATCGATACCACCATGGCCTGGACCGCTCTCCTGC-3’

Vl5/7 5’-AAATCGATACCACCATGGCCTGGACTCCTCTCCTTC-3’

Vl6/9 5’-AAATCGATACCACCATGGCCTGGGCTCCTCTCCTTC-3’

Vl3rm 5’-AAATCGATACCACCATGGCCTGGATCCCTCTCCTCC-3’

Vl3l 5’-AAATCGATACCACCATGGCCTGGACCCCTCTCTGGC-3’

Vl3e 5’-AAATCGATACCACCATGGCCTGGGCCACACTCCTGC-3’

Vl4c 5’-AAATCGATACCACCATGGCCTGGGTCTCCTTCTACC-3’

Vl8a 5’-AAATCGATACCACCATGGCCTGGATGATGCTTCTCC-3’

Cl2/7 5’-GATCGCGGCCGCTTATCAWGARCATTCTGYAGGGGCCACTG-3’

Oligonucleotide for construction of expression vector is:

Heavy chain expression vector:

VH1-L-NheI: 5’-GCGGCTAGCCACCATGGACTGGACCTGGAGG-3’

JH4/5-XhoI: 5’-GCGCTCGAGACGGTGACCAGGGTTCCCTG-3’

CHfw-XhoI: 5’-CGCGCTCGAGTGCCTCCACCAAGGGCCCATCGGTC-3’

CHrev-NotI:5’-GATCGCGGCCGCTTATCATTTACCCGGRGACAGGGAGAGGC-3’

Light chain expression vector:

VK1-L-NheI: 5’-GCGGCTAGCCACCATGGACATGAGGGTCCCY-3’

CK-NotI: 5’-GATCGCGGCCGCTTATCAACACTCTCCCCTGTTGAAGCTCTT-3’

EBV RT-PCR

In order to check potent proliferative response whether relevant to the existence of EBV, carry out EBV RT-PCR.RT step is described above.PCR condition is as follows: 94 DEG C of 7 minutes denaturing steps, is then 30 following circulations: 94 DEG C 30 seconds, 62 DEG C (HPRT1), 52 DEG C (LMP-1) and 58 DEG C (EBNA1/2) 30 seconds, 72 DEG C 30 seconds; Finally extend 7 minutes at 72 DEG C.Oligonucleotide for RT-PCR is as follows: HPRT1 forward (5`-TATGGACAGGACTGAACGTCTTGC-3`) and HPRT1 are oppositely (5 '-GACACAAACATGATTCAAATCCCTGA-3`); LMP-1 forward: (5`-GCGACTCTGCTGGAAATGAT-3`) and LMP-1 are oppositely (5`-GACATGGTAATGCCTAGAAG-3`); EBNA1/2 forward (5`-AGCAAGAAGAGGAGGTGGTAAG-3`) and EBNA1/2 are oppositely (5`-GGCTCAAAGTGGTCTCTAATGC-3`).

Except RT-PCR, we also directly carry out PCR on the cell precipitation be separated with QIAmp separating kit (Kai Jie company) and supernatant liquor DNA.

embodiment 1

Result

B cell phenotype

People's memory B cell depends on the ability of to cultivate and to test these cells within considerable time as the application of the platform of separate therapy medicine.Can cultivate in laboratory conditions and maintain human B cell, but the single B cell system being not enough to amplification, selection and cloning for antigen interested that holds time.We are according to the genetic modification exploitation immortalization technology to human B cell.We have studied the downstream targets of STAT5.A target spot is Bcl-6.The plasma cell differentiation that Bcl-6 can suppress B cell to be obstructed to propagation.The overexpression of Bcl-6 keeps the balance with BLIMP1, BLIMP1 to be the transcription factors stimulating its expression (by STAT3 onset) of the potent enhancing of B cell energy with IL-21.With regard to the growth that induction Ig produces cell (CD20-CD38+), BLIMP1 is required, and Bcl-6 can stop this process (cell keeps CD20 to express, so-called germinal center phenotype).

In order to study the time lag (skewing) that in B cell region, some cell mass is possible, carry out CFSE mark before stimulating fresh memory and primitive man's B cell to disclose, all cells start division, all B cell groups by etc. linked transduction (Fig. 2).Show with Bcl-6 transduction and the memory B cell cultivated under IL-21 and IL-4 exists.Constantly little 36, the levels of transduction of origi-nal B-cell is lower, and split speed is comparatively slow, but after cultivating 3 days again, identical with memory B cell (data do not show).

Next, we prove Bcl-6, and under Bcl-XL (the anti-apoptotic downstream targets of STAT5), CD40L intracellular signaling and IL-21 existence, (>3 month) the CD20+CD38dull phenotype (Fig. 3) of human IgG memory B cell can be maintained for a long time.

In addition, the phenotype of Bcl-6Bcl-XL B cell corresponds to activating B cell (such as, see table 1, the FACS dyeing of 3TT+B cell clone), because CD80, CD86 and HLA-DR high expression level in these cells.

The mensuration that three kinds of different B cl-6Bcl-XL B cell of cultivating together with IL-21 with CD40L intracellular signaling are cloned

Antibody membrane is expressed

Combine or κ and λ dyeing mensuration through antigen, the EBV negative cells of Bcl-6Bcl-XL transduction keeps BCR to express positive (Fig. 3 and 4).Therefore, this kind of cell is particularly suitable for separation and/or the specificity needed for screening after long-term cultivation, such as, utilize labelled antigen to screen, because this kind of cell will by its BCR in conjunction with described labelled antigen.With FACSAria, unicellular sorting is carried out to Bcl-6 and Bcl-XL of the TT marked in conjunction with PE two transduction B cell, thus verify.After three weeks, in 96 orifice plates, with suitable marker and TT-PE, the clone to unicellular sorting dyes, and measures combination (Fig. 4) with FACSCanto (BD).Conclusion is, needs B cell exists B-cell receptor, such as, in screening experiment, preferably by Bcl-6 and Bcl-XL transduction B cell, and infects without EBV.

Cell fission and growth curve

The B cell of Bcl-6Bcl-XL transduction on average divides 0.6 time every day.Division speed is different because of donor and culture cell density, and (Fig. 5 a).The division speed that anti-RSV clones D25 is every day 0.47 time (Fig. 5 b).Can lower than 1 cell/96 orifice plate culturing cell, for cloning object.

The antibody-secreting of Bcl-6Bcl-XL B cell

The B cell of Bcl-6Bcl-XL transduction on average secretes 1 μ g/ml antibody, and this is enough to the requirement (Fig. 6) of supplying preclinical study.Surprisingly, the antibody that the anti-RSV clone of D25 produces is three times of other test cell systems.

Measure EBV content

EBV RT-PCR is carried out to the mRNA of the Bcl-6Bcl-XL clone of cultivating together with IL-21 with CD40L intracellular signaling.In the clone obtained by this kind of immortalization technology, EBV genetic transcription thing (data do not show) do not detected.

Select step

Due to the stability that growth and BCR express, these cells are particularly suitable for being separated antigen-specific b cells.It gives us the chance using several different selection and cloning process.Be obtain an antigen-specific cellular immediately by the FACS of the antigen interested of mark or magnetic bead sorting after introducing Bcl-6 and Bcl-XL, thus improve the probability producing many antigen-specific b cells clone.Alternatively with memory (or any other) B cell that the Bcl-6Bcl-XL of low cell density (as 100 cells/well) Batch Culture purifying transduces.The supernatant liquor of these 100 cells/well cultures can be collected, and detect its specificity.Find that 100 cells/well cultures can identify antigen, then carry out subclone by limiting dilution culture, obtain monoclonal cell system.Use this two kinds of methods, we are the separable B cell clone to identifying Toxoid,tetanus (TT) more than 40.Therefore, the combination FACSAria according to TT and BCR selects these to clone, or screens a series of culture by ELISA and select, until be separated to single anti-TT monoclonal cell system (not shown).

The selection of RSV neutralizing antibody

Culture from 25 100 cells/well of donor B63 blocks rsv infection completely and copies.In and one of 100 cells/well cultures D10 produce potent anti-RSV antibodies, we are cultivated by limiting dilution and clone.One of monoclonal antibody D25 is used for follow-up study.Measure the monoclonal antibody D25 (Fig. 7) with IgG1 heavy chain and κ light chain through commercially available ELISA (Amsterdam San Kui company (Sanquin, Amsterdam), does not show) and effectively can block rsv infection, its IC ₅₀value is 0.5-1.5ng/ml (± 10pM), and the IC of clinical standard anti-RSV antibodies used (the beautiful pearl monoclonal antibody of handkerchief of Midi Miao Ni company exploitation) ₅₀be 0.453 μ g/ml (3.02nM) (H.Wu etc., 2005J.Mol.Biol. and A.Mejias etc., 2005Antimicrob.Agents Chemother.) (Fig. 8).

Antigen recognition

Except Neutralizing test, also measure the combination of D25 to the HEp2 cell of infection RSV.Produce scheme with conventional viral and infect HEp2 cell.Infect the HEp2 cell of RSV with tryptic digestion, and cultivate by 25-50 μ l culture supernatants.Washed cell, with Mouse-anti-human IgG-PE (BD or Jackson company (Jackson)) dyeing, to measure the combination of D25 antibody and cells infected.R-Biopharm ELISA control antibodies is used as interior mark.Fig. 9 a shows the combination of the complete HEp2 cell of D25 and infection RSV.

Because RSV coating (film) albumen comprises two kinds of protein, i.e. G-protein and F protein, so the combination detecting that D25 does not have VSV virus (the John K Rose friendship provides) cells infected of the false type of RSV F or RSV G-protein with use.As shown in figure 9b, D25 is potent is incorporated into the EL-4 cell infecting VSV-F albumen.When attempting the epi-position of research D25 and the identification of handkerchief beautiful pearl monoclonal antibody, hatch together with the D25 that the EL-4 cell of infection VSV-F albumen and consumption are increased progressively or the beautiful pearl monoclonal antibody of handkerchief.Washed cell, dyes with the mixture of 3 kinds of mouse-anti-RSV-F antibody (great Ke company (Dako)).Contrary with the handkerchief beautiful pearl monoclonal antibody that mouse-anti-RSV-F antibody competition combines the VSV-F cell infected, D25 is not in conjunction with uninfluenced (data show).

Fig. 9 c shows the beautiful pearl monoclonal antibody (Synagis) of handkerchief and D25 is incorporated into the HEp2 cell of infection with concentrationdependent manner.Due to these two kinds of antibody all with 1:1 in conjunction with its target protein, so there is no difference with the combination of HEp2 cell infected.

RSV antigen combines and neutralizes the frequency of cloning

We calculate, and are 17% for donor B63 in conjunction with the frequency of the antigen specific memory B cell of RSV, in and the frequency of antigen-specific cellular of RSV be 6%.The conformational epitope that D25 combines is different from the epi-position of handkerchief beautiful pearl monoclonal antibody identification.This point can as can be seen from Figure 10, and wherein D25 does not combine the sex change linear epitope of the rsv infection cell lysate submission of the cracking be coated on ELISA flat board, and the beautiful pearl monoclonal antibody of handkerchief is incorporated into sex change (F) albumen.

The abstraction and purification of antibody fragment

We from some B cell systems, can comprise in high RSV and clone the volume of D25 acquisition up to 500ml.These culture supernatants contain at least 2 μ g/ml, and therefore, we should be able to obtain enough antibody purifications, with carry out clinical before (animal) research.With montage (Montage) antigen purification test kit (Millipore Corp. (Millipore blocked in Massachusetts, United States Bill, Billerica, MA, USA)) and HiTrap a-protein HP post (Belgium is for GE health care company (the GE Healthcare of root, Diegem, Belgium)) carry out purifying.

In addition, by Lipofectamine LTX (hero company), heavy chain and the light chain transfection 293T cell of the D25 of subclone in pCDA3.1 protein expression vector is utilized.IgG content in supernatant liquor is about 22 μ g/ml (cumulative volume 50ml).This kind of antibody of the nucleotide sequence generation of the D25B expression of cell lines antibody of clone also identifies the HEp2 cell (data do not show) of infection.

Antibody sequence

Figure 11 a shows heavy chain and light chain nucleotide and the aminoacid sequence of B63D10-D25 clone.The RT-PCR of use standard and antibodies specific primer, measure heavy chain (Vh1-69) and light chain (VkI O8/O18) sequence.Use the whole antibody sequence of TOPO carrier cloning, sequence is subcloned in pCDNA3.1 mammalian proteins expression vector (hero company) after controlling.Figure 11 b and 11c describes VH and the VL4 chain of this clone, and asterisk represents and the sudden change compared with Vh1-69 Germline sequences that must occur between Affinity maturation and further B cell selecting period.

In a word, we show both and utilize transgenosis Bcl-6 to be separated people's memory B cell with Bcl-XL, characterize and long-term cultivation.They are that we are separated the antibody with unique property, such as the necessary instrument of anti-RSV monoclonal antibody B63D10-B25.Because B cell is originated from people, so they are not difficult to be developed to curative drug.

embodiment 2

As previously mentioned (p44 ' antibody sequence '), D25 heavy chain and light chain are cloned in standard expression vectors.Can the expression constructs of at utmost marking protein in order to produce, carry out codon optimized by GENEART (Regensburg, Germany (Regensburg, Germany)) to D25 heavy chain and sequence of light chain.In this approach, produce extra restriction site, to simplify cloning process in the future, but the most important thing is, optimize the nucleotide codon translating into aminoacid sequence, at utmost to translate into protein.Therefore, nucleotide sequence is optimized but aminoacid sequence remains unchanged.Example 4 shows the D25 of the B cell supernatant liquor being derived from purifying, the neutralising capacity of D25 that restructuring D25 and GENEART optimizes.They can effectively in and RSV.

Compared with original D25 sequence, Figure 12 is shown in the modification that GENEART does.

embodiment 3

After having carried out external RSV Neutralizing test, we have detected D25 monoclonal antibody in model in vivo.Once recording the model being used for anti-RSV test in body is BALB/c mouse and cotton mouse (Sigmodon hispidus) (Mejias A etc., Antimicrobial Agents and chemotherapy 2004; 1811st page, Johnson S etc., JID 1997; 1215th page and Wu H etc., JMB 2007: the 652 pages).Clearly, BALB/c mouse model is the most weak model, but is difficult to acquisition due to cotton mouse and raises, and first we set up D25 test in BALB/c mouse.

Scheme: the RSV specific antibody in BALB/c, 5 days

Experimental design:

Within-1 day .I.P. injects 100 μ l antibody

Within 0th day .I.N infects 1x10 ⁷pfu RSV A2 (50 μ l)

1-5 days, checks the general health of mouse and weighs

5th day, become celestial, collect BAL, blood and lung

Pass through venipuncture

2.0ml BAL is collected by tracheae

Collect lung

Immediately TCID is started to BAL material (1ml) ₅₀.

1ml BAL material (ELISA cytokine/RT-PCR) is frozen in-80C

TCID is carried out to the lung material (1ml) of preparation ₅₀

1ml lung material (ELISA cytokine/RT-PCR) is frozen in-80C

Collection/centrifugal blood, carries out hIgG ELISA to serum, is stored in-80C

The results are shown in Figure 13:

(A) RSV stimulation (1x10 is carried out by nose spraying ⁷rSV-A2 particle) the day before yesterday, animal IP is injected to Synagis (Midi Miao Ni company), purifying D25 or the IgG1 control antibodies (excellent Lycra company (Eureka)) (table 3) of different amount.(Figure 13 B) measured the human IgG level in mice serum from the 5th day, and antibody serum level declined in 5 days; Table 4 shows transformation period general introduction.The virus titer that Figure 13 D describes treatment in the 5th day and do not treat in the lung-douching fluid (BAL) of animal, and Figure 13 E describes treatment and do not treat peripheral blood T and the B cell quantity of mouse.The lung of animal is not treated and is treated in Figure 13 F display and segmental bronchus infiltrates the tissue slice of (usually mainly eosinocyte).

Conclusion/result:

Calculate according to (linearly) and estimate that the D25 transformation period is 5-9 days: injection 60 and 30 μ g antibody (be respectively 2 and 1mg/kg) in the 0th day, 33 or 16 μ g (cumulative volume 1,5 of mouse) detected at the 5th day.When with the 0th day, every animal injection 0,5mg/kg started, drop to 11 μ g the 5th day Ig level from 15 μ g, this shows that the transformation period is 9 days (table 4).

Table 4

TCID ₅₀the virus titer of measuring shows, can 1x10 be detected in control animal ⁴pFU, and can't detect virus in the animal treated at Synagis (2mg/kg) or D25 (2,1 and 0,5mg/kg).

Higher peripheral blood cd4 t cell and B220B percentage of cells is maintained with the animal of Synagis or D25 treatment.The animal that the %CD4T cell of the animal that Synagis (2mg/kg) treats is treated lower than D25.Although this may not significantly, it should be noted that and keep high-caliber B and T cell with the animal capable that low dosage D25 (1 and 0,5mg/kg) treats compared with randomized controlled treatment animal.

Although histological data (Figure 13 F) is not quantitative data, can find out that Synagis and D25 reduces immunocyte in lung and peribronchial interior stream compared with the control.Relatively during D25 and Synagis, less with peribronchial situation in cellular infiltration to lung in the animal of D25 treatment.

In order to detect D25 in cotton mouse, set up the animal of testing and comparing with Synagis and D25 pretreat before RSV-X virus stimulates at NVI (Holland is than Dove (Bilthoven, Netherlands)).

embodiment 4

Except B63-D10-D25, we are separated to three kinds of new potent RSV neutralizing antibodies (AM14, AM16 and AM23) by same donor (B63).By initial according in RSV and to select and the frozen 100 cells/well batch B cell cultures in liquid nitrogen thaw, detect culture supernatants to the combination of HEp2 cell infecting RSV.We detect and the combination of Hep2 cell infected, because its natural oligomerization RSV membranin that is antibody recognition is as the marker of F and G-protein, and can be used as the good predict index that neutralizes.Detection in conjunction with time, single cell culture is carried out to cell, screening combine to obtain clone.By all three kinds of antibody clonings in the GENEART carrier being initially D25 structure.In addition, the antibody such as such as D25 can identify RSV-F albumen (not shown).In 293T cell after cloning and expressing, purification of recombinant proteins (Nucleotide and aminoacid sequence are shown in Figure 14 A, B and C).Vero and HEp2 cell detects antibody in several main RSV isolate and (Figure 15).All three kinds of antibody are IgG1 isotype.AM14 has κ light chain, and AM16 and AM23 has lambda light chain.All three kinds of antibody as D25 in its antibody variable region all containing somatic hypermutation, this experiences Affinity maturation during pointing out their germinal center reactions in vivo, and this is the process producing unique antibodies sequence.

The results are shown in Figure 15-I and 15-II: fasten the D25 (sD25) of clear liquid, D25 (rD25), the codon optimized D25 (rD25GA) of restructuring GENEART of restructuring purifying, AM14, AM16, AM23 (being the recombinant protein of purifying) and Synagis by the B cell being derived from purifying and carry out the viral Neutralizing test of RS.On two kinds of different clones (Figure 15-I) Vero and (Figure 15-II) Hep2 cell, the antibody neutralising capacity of virus is detected: A2 (A), X (B) and 2006/1 (C) are RSV hypotype A, and viral Z (D) and 2007-2 (E) is hypotype B by different antibodies.By 100TCID ₅₀often kind of virus add in the antibody-solutions with DMEM/1%FCS serial dilution, cultivate 1 hour, then add 100ul Vero or HEp2 cell (1x10 for 37 DEG C ⁶/ ml).Do not wash virus-antibody mixture off.After three days, remove supernatant liquor, at room temperature use 80% acetone fixed cell 10 '.After removing acetone, dry fixing cellular layer, keeps 4 DEG C or frozen in-20 DEG C.In order to the HEp2 cell dyeing to infection RSV, first with 5% milk powder blocking of plates of PBS 0.1% polysorbas20 preparation, then dull and stereotyped 3 times of washing, then at 37 DEG C and the anti-RSV-HRP (1:500 of polyclonal goat, the biological design corporation of maine state of u.s.a Mermithidae) cultivate 3-5 hour together, fully wash.Next, at room temperature institute porose 30 ' is hatched with AEC substrate.The stove point infected dyes redness, counts by opticmicroscope visual inspection.

Result/conclusion

All antibody can in and RSV A and B strain (table 5).Usually, with RSV virus during different D25 antibody capable is effective, but can be observed difference between less experiment.AM14 and D25 effect is identical, and AM16 and Synagis effect is identical.But, AM23 can effectively in and RSV A strain, but in it and the effect of RSV B strain poor, but still suitable with Synagis.

Table 5 IC50 value (ng/ml)

On Vero or HEp2 cell, the IC50 value of often kind of antibody to RS virus subtype A is calculated as average 50% neutralization value to three kinds of virus strain (A2, X and 2006-1).On Vero or HEp2 cell, the IC50 value of often kind of antibody to RS virus subtype B is calculated as average 50% neutralization value to two-strain strain (2007-2 and Z).Carry out each Neutralizing test in triplicate, and repeat twice (being also shown in Figure 15 A and B).

The culture supernatants that sD25=purified splenic B cell produces

The restructuring D25 of rD25=purifying

RD25GA=293T cell conditioned medium liquid and the codon optimized restructuring D25 of GENEART

embodiment 5

Collaborative and the blocking effect of anti-RSV antibodies

Whether interfere with each other identification to RSV F protein to analyze D25, Synagis or new AM antibody group, we hatch the HEp2 cell of infection RSV, until they reach maximum combined platform in advance with the unmarked antibody of increasing concen-trations.We are often kind of antibody determination plateau, and combining when plateau Ig amount increases no longer increases.(not shown).After washing, hatch together with the Synagis that sample and D25 or APC that the PE of standard dose (3pmol) marks are marked.This dosage also reaches maximum combined.

Result

As shown in figure 16, when infecting the HEp2 cell of RSV with unlabelled Synagis or D25 preincubate, the Bound moisture pancake of Synagis and D25 of mark and these cells is low.And Synagis can reduce the combination of AM16 induction slightly.D25 combines and is strongly blocked by AM23, but by contrast, combines with the D25 of enhancing strong after AM14 preincubate.This shows that the epi-position that D25 identifies does not expose usually completely, but AM14 strengthens it and exposes after being combined with its natural epitopes.This proves that these two kinds of antibody can play a role together and strengthen neutralizing effect.

brief Description Of Drawings

Fig. 1

The separation of human IgG positive memory B cells.Utilize phenanthrene to hatch together with anti-CD22 magnetic bead by the PBMC that is separated from dark yellow layer of density fractionation (An Ma West Asia company), then use MACS post (close special company) to be separated.Then, CD22 positive cell is hatched together with the antibody (BD) of IgA with people CD19, CD27, IgM, IgD.With the cell that the sorting of high speed unicellular sorting technology (FACSAria, BD) is IgM, IgD, IgA feminine gender and CD19, CD27 positive.

Fig. 2

CFSE dyes.Be separated Freshman memory B cell, with CSFE mark, stimulate 36 hours with IL-21, then transduce with Bcl-6-IRES-NGFR.Cell and IL-21 hatch 3 days more jointly, then measure CFSE content.CFSE dyestuff is diluted during each cell fission.

Fig. 3

With Bcl-6 and Bcl-XL or only with the example of the human B cell of Bcl-XL transduction.Cell is being maintained on the expression CD40L of radiation and the L cell of cytokine IL-21.Left figure is shown the BCR measured by κ and λ dyeing and expresses (the κ λ positive cell of 93% is IgG isotype, does not show).The X-axis of right figure represents that CD38 expresses, and Y-axis represents that CD20 expresses.CD38 ^dullcD20 ⁺dyeing is shown to be memory or Germinal center B cell; CD38 ⁺cD20 ^-dyeing is shown to be plasmablast.

Fig. 4

The separation of the antigen-specific human B cell of immortalization.Be separated people's memory B cell as shown in Figure 1, subsequently with Bcl-6-IRES-NGFR and Bcl-XL-IRES-GFP transduction.Expression of NGF R, GFP is separated and the cell of the tetanus toxin that can mark in conjunction with PE-with FACSAria.In 96 hole flat-bottomed plates, carrying out single cell culture to cell under the L cell and IL-21 existence of radiation, then utilizing FACS Canto (BD) to combine according to TT-PE and selecting.

Fig. 5

The accumulation Growth of Cells of 6XL B cell clone and division speed.To clone from the anti-TT of (A) two and the B cell of (B) anti-RSV clone (B63D10-D25) at IL-21 and cultivating under the existence of the L cell of radiation.

Fig. 6

Contain the IMDM of 8%FCS and green grass or young crops/Streptomycin sulphate with 1,0ml, with 200,000 cell/24 orifice plate starts to cultivate fresh culture thing.FCS used is normal (extra large cloning companies (HyClone)) or ultralow ox IgG FCS (Ji Buke company (Gibco)).After 3 days, change culture supernatants, cell quantity is adjusted to 200,000 cells/ml.Show the average IgG output in 3 days that measure on 3 continuous time points, there is no significant difference (p value is 0.2).

Fig. 7

In order to determine the light chain phenotype of the anti-RSV clone of D25, with κ-phycoerythrin or λ-phycoerythrin (BD) antibody, D25B clone is dyeed.Only have κ-this clone of phycoerythrin antibodies, show that this antibody has κ light chain.

Fig. 8

The 100 cells/well cultures from donor B63 are cultivated with Bcl-6Bcl-XL positive human memory B cell.One of them culture D10 demonstrates strong neutralizing effect.Preparation is derived from the monoclonal cell system of LD, with RSV A-2 virus during a D25 is effective.There is shown comparing of D25 and handkerchief beautiful pearl monoclonal antibody (synagis) and the anti-RSV of polyclonal goat.Do not show the irrelevant culture supernatants of the B cell clone of the Bcl6Bcl-XL transduction of cultivating with IL-21 and CD40L intracellular signaling, it can produce high-level antibody, but does not block rsv infection.D25 clone is used for further sign.

Fig. 9

In fig. 9 a: with the density of 10-12e6 cell/T175 culturing bottle (Nunc), HEp2 cell is inoculated with IMDM/5%FCS.Second day, replace substratum with 5ml containing the substratum of RSV virus (1.0MOI), incubate at room temperature 45 ', then added 20ml fresh culture, 37 DEG C of o/n culturing cells.3rd day, replace substratum with IMDM/1%FCS, cover tightly lid 37 DEG C of o/n and cultivate.4th day, use trypsin treatment with PBS washed cell.In order to the cell infected that dyes, tentatively hatch by culture supernatants.Carry out second time with anti-human igg-PE (BD) to hatch.With LSRII (BD) analysis of cells.As positive control, use the positive control of commercial ELISA Assay kit (r-Biopharm).

In figure 9b: infect EL-4 cell by the VSV virus (John Rose friendship provides) with RSV F or the false type of G-protein, and cultivate by D25 culture supernatants.Washed cell, cultivates with Anti-Human IgG-PE (Jackson company), to measure the combination of D25 and cells infected.D25 and the combination of VSV virus infected cell with the false type of RSV F protein only detected.

Fig. 9 c shows the beautiful pearl monoclonal antibody (Synagis) of handkerchief and D25 is incorporated into the HEp2 cell of infection with concentrationdependent manner.That show is average fluorescent strength (MFI).

Figure 10

The combination of the HEp2 cells infected lysate of the beautiful pearl monoclonal antibody (synagis) of the anti-RSV of polyclonal goat (positive control), handkerchief and D25 and bag quilt.

Figure 11

The sequential analysis of D25 clone.11a shows heavy chain and the Nucleotide of variable region of light chain and the aminoacid sequence of prediction.11b/c display is D25 heavy chain and sequence of light chain compared with prediction germline.Asterisk expresses possibility in the sudden change selected and occur in affine ripening process in body that B cell is cloned.

Figure 12

The B cell system cloning and expressing recombinant human antibody of being transduceed by BCL6BCL-xL.In D25 antibody related content, recorded this process (Figure 11).There is shown the GENEART nucleotide modification compared with original D25 sequence, should be noted that these sudden changes do not change the amino acid composition of D25 antibody.

Figure 13 A-13F

BALB/c mouse is stimulated with D25 and Synagis of the B cell supernatant liquor being derived from purifying.(A) RSV stimulation (1x10 is carried out by nose spraying ⁷rSV-A2 particle) the day before yesterday, animal IP is injected to Synagis (Midi Miao Ni company), purifying D25 or the IgG1 control antibodies (excellent Lycra company (Eureka)) (table 3) of different amount.(B) from the 5th day, measure the human IgG level in mice serum, antibody serum level declined (C) in 5 days; Table 4 shows transformation period general introduction.The virus titer that Figure 13 D describes treatment in the 5th day and do not treat in the lung-douching fluid (BAL) of animal, and Figure 13 E describes treatment and do not treat peripheral blood T and the B cell quantity of mouse.(F) lung of animal is not treated and treated to display and segmental bronchus infiltrates the tissue slice of (usually mainly eosinocyte).

Figure 14 A-14C

The Nucleotide of three kinds of new potent RSV neutralizing antibody (A) AM14, (B) AM16 and (C) AM23 and aminoacid sequence.

Figure 15-I and Figure 15-II

Fasten the D25 (sD25) of clear liquid, D25 (rD25), the codon optimized D25 (rD25GA) of restructuring GENEART of restructuring purifying, AM14, AM16, AM23 (being the recombinant protein of purifying) and Synagis by the B cell being derived from purifying and carry out the viral Neutralizing test of RS.On two kinds of different clones (Figure 15-I) Vero and (Figure 15-II) Hep2 cell, the antibody neutralising capacity of virus is detected: A2 (A), X (B) and 2006/1 (C) are RSV hypotype A, and viral Z (D) and 2007-2 (E) is hypotype B by different antibodies.By 100TCID ₅₀often kind of virus add in the antibody-solutions with DMEM/1%FCS serial dilution, cultivate 1 hour, then add 100ul Vero or HEp2 cell (1x10 for 37 DEG C ⁶/ ml).

Figure 16

The rD25 of Synagis with the PE mark of the APC mark of fixed amount (3pmol) and the relative combination of the HEp2 cell of infection RSV, unmarked antibody preincubate shown in HEp2 cell increasing concen-trations.

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Claims

1. be separated, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its Functional portions, derivative and/or an analogue, it comprises:

2. antibody as claimed in claim 1, Functional portions, derivative or analogue, it is characterized in that, its sequence of heavy chain comprises the sequence identical with sequence QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLG TVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHY FDN WGQGTLVTVSS at least 70%, and/or its sequence of light chain is identical with sequence D IQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETG VPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTV at least 70%.

3. an antibody, or the Functional portions of described antibody, derivative and/or analogue, its infect RSV HEp-2 cell external Neutralizing test in IC ₅₀value is less than 10ng/ml, is preferably less than 2ng/ml.

4. be separated, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its Functional portions, derivative and/or an analogue, it comprises:

-heavy chain CDR1 sequence, it comprises the sequence identical with sequence GFSFSHYA at least 80%, and/or

-heavy chain CDR2 sequence, it comprises the sequence identical with sequence ISYDGENT at least 80%, and/or

-heavy chain CDR3 sequence, it comprises the sequence identical with sequence A RDRIVDDYYYYGMDV at least 80%, and/or

-light chain CDR1 sequence, it comprises the sequence identical with sequence QDIKKY at least 80%, and/or

-light chain CDR2 sequence, it comprises the sequence identical with sequence D AS at least 80%, and/or

-light chain CDR3 sequence, it comprises the sequence identical with sequence QQYDNLPPLT at least 80%.

5. antibody as claimed in claim 4, Functional portions, derivative or analogue, it is characterized in that, its sequence of heavy chain comprises the sequence identical with sequence EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGE NTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDV WGQGATVTVSS at least 70%, and/or its sequence of light chain is identical with sequence D IQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETG VPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGGTKVEIKRTV at least 70%.

6. be separated, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its Functional portions, derivative and/or an analogue, it comprises:

-heavy chain CDR1 sequence, it comprises the sequence identical with sequence GFTFSSYN at least 80%, and/or

-heavy chain CDR2 sequence, it comprises the sequence identical with sequence ISAGSSYI at least 80%, and/or

-heavy chain CDR3 sequence, it comprises the sequence identical with sequence A REDYGPGNYYSPNWFDP at least 80%, and/or

-light chain CDR1 sequence, it comprises the sequence identical with sequence SSNIGAGYD at least 80%, and/or

-light chain CDR2 sequence, it comprises the sequence identical with sequence GNT at least 80%, and/or

-light chain CDR3 sequence, it comprises the sequence identical with sequence HSYDRSLSG at least 80%.

7. antibody as claimed in claim 6, Functional portions, derivative or analogue, it is characterized in that, its sequence of heavy chain comprises the sequence identical with sequence EVQLVETGGGLAQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSHISAGSS YIYYSDSVKGRFTVSRDNVRNSVYLQMNSLRAADTAVYYCAREDYGPGNYYSPNWF DPWGQGTLVTVSS at least 70%, and/or its sequence of light chain is identical with sequence QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNR PSGVSDRFSGSKSGTSASLAITGLQAEDEADYYCHSYDRSLSGSVFGGGTKLTV at least 70%.

8. be separated, synthesis or restructuring can the antibody of specific binding respiratory syncytial virus or its Functional portions, derivative and/or an analogue, it comprises:

-heavy chain CDR1 sequence, it comprises the sequence identical with sequence GFNFHNYG at least 80%, and/or

-heavy chain CDR2 sequence, it comprises the sequence identical with sequence VWYDGSKK at least 80%, and/or

-heavy chain CDR3 sequence, it comprises the sequence identical with sequence VRDKVGPTPYFDS at least 80%, and/or

-light chain CDR1 sequence, it comprises the sequence identical with sequence NIGSET at least 80%, and/or

-light chain CDR2 sequence, it comprises the sequence identical with sequence D DD at least 80%, and/or

-light chain CDR3 sequence, it comprises the sequence identical with sequence QVWDRSNYHQV at least 80%.

9. antibody as claimed in claim 6, Functional portions, derivative or analogue, it is characterized in that, its sequence of heavy chain comprises the sequence identical with sequence EVQLVESGGNVVKPGTSLRLSCAATGFNFHNYGMNWVRQAPGKGLEWVAVVWYDGS KKYYADSVTGRFAISRDNSKNTLYLQMNSLRVEDTAVYYCVRDKVGPTPYFDSWGQ GTLVTVSS at least 70%, and/or its sequence of light chain is identical with sequence SYVLTQPPSVSLAPGGTAAITCGRNNIGSETVHWYQQKPGQAPVLVVYDDDDRPSG IPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSNYHQVFGGGTKLTV at least 70%.

10. antibody, Functional portions, derivative or analogue as claimed in any one of claims 1-9 wherein, it is characterized in that, they are people's antibody and/or chimeric antibody.